Hybridization assay probes and methods for detecting the presence of neisseria meningitiidis subtypes A, C and L in a sample

ABSTRACT

The present invention discloses hybridization assay probes, amplification primers, nucleic acid compositions and methods useful for detecting Neisseria nucleic acids. Hybridization assay probes and amplification primers that selectively detect  Neisseria meningitidis  and distinguish those  Neisseria meningitidis  from  Neisseria gonorrohoeae  are disclosed. Other hybridization probes selectively detect  Neisseria gonorrohoeae  and not  Neisseria meningitidis  are also described.

This application is a continuation of application Ser. No. 08/962,369,filed Oct. 31, 1997, now U.S. Pat. No. 6,100,027, which is acontinuation of application Ser. No. 08/484,607, filed Jun. 7, 1995, nowU.S. Pat. No. 5,747,252.

FIELD OF THE INVENTION

The inventions described and claimed herein relate to the design and useof amplification oligonucleotides and nucleic acid probes to Neisseriagonorrhoeae and Neisseria meningitidis which allow detection of theseorganisms in test samples.

BACKGROUND OF THE INVENTION

The genus Neisseria includes two gram-negative species of pyogenic coccithat are pathogenic for man, and that have no other known reservoir: themeningococcus (Neisseria meningitidis) and the gonococcus (Neisseriagonorrhoeae). A number of non-pathogenic species also inhabit the upperrespiratory tract of humans and may be easily confused withmeningococci. Meningococcal meningitis was recognized as a contagiousdisease early in the 19th century and is especially prevalent amongmilitary personnel. The causative agent of meningococcal meningitis isNeisseria meningitidis.

Neisseria gonorrhoeae is one of the main causes of epidemic sexuallytransmitted disease and is prevalent in the United States. Infectionwith Neisseria gonorrhoeae causes many, common symptoms includingurethritis, cervicitis, and proctitis. In addition, chronic infectionwith Neisseria gonorrhoeae can cause pelvic inflammatory disease.meningococci have polysaccharide-containing capsules. Gonococcis mayalso possess capsules, but the exact chemical composition of such acapsule is unknown. In addition, both gonococci and meningococci mayhave pili which play a role in virulence.

Meningococci and gonococci are difficult to cultivate and requirespecial techniques to grow the organisms from body fluids. In addition,selective culture medium, (for example, Thayer-Martin medium) and growthin 3-10% carbon dioxide at approximately 35° C. is required to maximizethe culture of organisms.

In addition to the difficult cultivation, the gonococcus andmeningococcus detection by immunoassay suffers a lack of sensitivity andspecificity. This appears to be due to the cross reaction betweenvarious other pathogens and non-pathogenic microorganisms often found inthe same clinical specimens.

Oligonucleotides for the amplification of nucleic acid for detection ofNeisseria have been described. Biken-meyer and Armstrong, J. Clin.Microbiol. 30:3089-3094 (1992), describe probe sets for use in theligase chain reaction directed to the Opa and pilin genes of Neisseriagonorrhoeae. Kristiansen et al. Lancet 340:1432-1434 (1992) describeprimers directed to an insertion element referred to as IS1106 foramplification and detection of Neisseria meningitidis. McLaughlin etal., Mol. and Cell Probes 7:7-17 (1993) describe primers for use in thepolymerase chain reaction directed to the 16S-23S rRNA internaltranscribed spacer and a set of primers directed to a subregion of the16S rRNA of Neisseria meningitidis. Probes for the detection of rRNA orrDNA sequences of Neisseria gonnorhoeae and/or Neisseria meningitidishave been described by Granato and Franz J. Clin. Microbiol. 28:944-948,(1990), Wolff, U.S. Pat. No. 5,173,401 (Dec. 22, 1992), Rossau and VanHeuverswijn, European Patent Application Publication No. 0 337 896,Hogan et al. PCT/US87/-03009, and Barns et al., U.S. Pat. No. 5,217,862(Jun. 8, 1993).

SUMMARY OF INVENTION

The featured invention discloses and claims novel and usefulamplification oligonucleotides, helper oligonucleotides, andoligonucleotide hybridization assay probes which are designed to becomplementary to specific regions of the rRNA (ribosomal RNA) or rDNA(ribosomal DNA) nucleotide sequences of Neisseria, or oligonucleotideshaving a nucleic acid sequence substantially corresponding to a specificportion of Neisseria rRNA or rDNA nucleotide sequence or its complement.Because these amplification oligonucleotides, helper oligonucleotidesand hybridization assay probes are derived from the rRNA of pathogenicNeisseria, a superior detection assay is obtained due to the higherlevel of RNA expressed from these rRNA genes and the lack of lateraltransfer of the rRNA sequences between organisms.

The amplification oligonucleotides and oligonucleotide hybridizationassay probes function by hybridizing to target Neisseria 16S and 23SrRNA and/or rDNA gene sequences under stringent hybridization assayconditions. In preferred embodiments, the probes and amplificationoligonucleotides described herein, when used together, can distinguishNeisseria meningitidis from other microorganisms found in clinicalsamples such as blood or tissues and from Neisseria gonorrhoeae species.Accordingly, the amplification oligonucleotides and hybridization assayprobes may be used in an assay to specifically detect and/or amplifyNeisseria meningitidis-derived nucleic acids. In preferred embodiments,the hybridization assay probes described herein are able to selectivelyhybridize to nucleic acids from Neisseria meningitidis over those fromNeisseria gonorrhoeae under stringent hybridization conditions. In someembodiments of the present invention, the hybridization assay probecomprises an oligonucleotide that contains a reporter group such as anacridinium ester or a radioisotope to help identify hybridization of theprobe to its target sequence. In some embodiments of the presentinvention, the amplification oligonucleotide optionally has a nucleicacid sequence recognized by an RNA polymerase or which enhancestranscription initiation by an RNA polymerase.

The present invention features hybridization assay probes useful fordetecting the presence of nucleic acids from Neisseria. Preferably, thehybridization assay probes are selected from the following nucleotidesequences:

SEQ ID NO 11: GGCTGTTGCT AATATCAGCG

SEQ ID NO 12: GGCTGTTGCT AATACCAGCG

SEQ ID NO 15: CGCTGATATT AGCAACAGCC

SEQ ID NO 16: CGCTGGTATT AGCAACAGCC

SEQ ID NO 25: GGCUGUUGCU AAUAUCAGCG

SEQ ID NO 26: GGCUGUUGCU AAUACCAGCG

SEQ ID NO 27: CGCUGAUAUU AGCAACAGCC

SEQ ID NO 28: CGCUGGUAUU AGCAACAGCC

SEQ ID NO 1: GAACGTACCG GGTAGCGG

SEQ ID NO 3: GCCAATATCG GCGGCCGATG

SEQ ID NO 29: CCGCTACCCG GTACGTTC

SEQ ID NO 30: CATCGGCCGC CGATATTGGC

SEQ ID NO 31: GAACGGCCGC CGATATTGGC

SEQ ID NO 32: GCCAAUAUCG GCGGCCGAUG

SEQ ID NO 33: CCGCUACCCG GUACGUUC

SEQ ID NO 34: CAUCGGCCGC CGAUAUUGGC

The present invention features hybridization assay probes useful fordetecting nucleic acids from Neisseria meningitidis. These hybridizationassay probes are preferably selected from the following nucleotidesequences:

SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG

SEQ ID NO: 12 GGCTGTTGCT AATACCAGCG

SEQ ID NO: 15 CGCTGATATT AGCAACAGCC

SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC

SEQ ID NO: 25 GGCUGUUGCU AAUAUCAGCG

SEQ ID NO: 26 GGCUGUUGCU AAUACCAGCG

SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC, and

SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC.

The present invention also features hybridization assay probes usefulfor detecting Neisseria gonorrhoeae nucleic acids. Preferably, thesehybridization assay probes have a nucleotide sequence selected from oneof the following nucleotide sequences:

SEQ ID NO: 1: GAACGTACCG GGTAGCGG

SEQ ID NO: 29: CCGCTACCCG GTACGTTC

SEQ ID NO: 30: CATCGGCCGC CGATATTGGC

SEQ ID NO: 31: GAACGUACCG GGUAGCGG

SEQ ID NO: 32: GCCAAUAUCG GCGGCCGAUG

SEQ ID NO: 33: CCGCUACCCG GUACGUUC

SEQ ID NO: 34: CAUCGGCCGC CGAUAUUGGC

Another aspect of the present invention is a probe mix comprising ahybridization assay probe of the present invention together with ahelper oligonucleotide (probe). Preferably, helper oligonucleotides areused to facilitate the specific hybridization of the assay probe to itstarget nucleic acid; helper oligonucleotides are described by Hogan andMilliman U.S. Pat. No. 5,030,557 which is hereby incorporated byreference and enjoys common ownership with the present invention.Oligonucleotides used as helper probes in this invention include thefollowing sequences:

SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG

SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC TACGTG

SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG

SEQ ID NO: 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG

SEQ ID NO: 13 GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA

SEQ ID NO: 14 GCTGATGACG GTACCTGAAG AATAAGCACC GGC

SEQ ID NO: 35 GCCUUCGGGU UGUAAAGGAC UUUUGUCAGG GAAGAAAA

SEQ ID NO: 36 GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC

SEQ ID NO: 17 TTTTCTTCCC TGACAAAAGT CCTTTACAAC CCGAAGGC

SEQ ID NO: 18 GCCGGTGCTT ATTCTTCAGG TACCGTCATC AGC

SEQ ID NO: 37 UUUUCUUCCC UGACAAAAGU CCUUUACAAC CCGAAGGC, and

SEQ ID NO: 38 GCCGGUGCUU AUUCUUCAGG UACCGUCAUC AGC

Another aspect of the present invention includes compositions fordetecting Neisseria meningitidis and Neisseria gonorrhoeae that arenucleic acid hybrids formed between an oligonucleotide of the presentinvention and a specific region of a nucleotide polymer from a Neisseriameningitidis or Neisseria gonorrhoeae. Generally, the nucleotide polymercontains a nucleic acid sequence that substantially corresponds to anoligonucleotide sequence of the present invention or its complement andis derived from the rRNA or the rDNA encoding the ribosomal RNA of theNeisseria meningitidis or Neisseria gonorrhoeae. The oligonucleotidepresent in these compositions may be an amplification oligonucleotide, ahelper oligonucleotide, a hybridization assay probe, or a combinationthereof. Thus, compositions of the present invention may contain one ormore amplification oligonucleotides, one or more helperoligonucleotides, and one or more hybridization assay probes.

The compositions of the present invention containing a probe hybridizedto its target sequence are useful for detecting the presence of anucleic acid sequence. Compositions of the present invention containinga helper oligonucleotide hybridized to its target nucleic acid sequenceare useful for making a particular portion of the target nucleic acidavailable for hybridization. Compositions of the present inventioncontaining an oligonucleotide primer hybridized to its target sequenceare useful for creating an initiation site for a polymerase at the 3′end of the primer, and/or providing a template for extension of the 3′end of the target sequence.

The present invention also contemplates methods for detecting thepresence of Neisseria in which a test sample is contacted with a nucleicacid hybridization assay probe under stringent hybridization assayconditions wherein the nucleic acid hybridization assay probe is capableof hybridizing to Neisseria meningitidis target nucleic acid sequencesand not to the nucleic acid sequences from Neisseria gonorrhoeae. Thepresent invention also contemplates oligonucleotides and the equivalentsthereof used in these methods that optionally contain a reportermolecule that aids in the identification of the hybridization of theprobe to its target sequence. This invention is useful for detecting thepresence of Neisseria nucleic acids in test samples from humans such asblood, blood derived samples, tissues, tissue derived samples, otherbody fluids and body samples.

The present invention also contemplates methods for detecting thepresence of Neisseria meningitidis in which the nucleic acid isamplified using at least one amplification oligonucleotide of thepresent invention. In preferred embodiments, that amplification is thenfollowed by a detection step in which the amplified nucleic acid isdetected using an oligonucleotide hybridization assay probe of thepresent invention. The methods of the present invention also contemplatethe use of amplification oligonucleotides which include the nucleotidesequence for an RNA promoter.

In another aspect, the invention features amplification oligonucleotidesuseful for detection of organisms of the genus Neisseria in anamplification assay. Such oligomers preferably substantially correspondto one of the following nucleotide sequences:

SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC

SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA GTAACATA

SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG

SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG

SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT

SEQ ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC

SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC

SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA

SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG

SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG

SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU, and

SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC

where the oligomer may be unmodified or contain a modification such asaddition of a specific nucleic acid sequence to 5′ terminus that isrecognized by an RNA polymerase, (including but not limited to thepromoter sequence for T7, T3, or SP6 RNA polymerase), and/or sequenceswhich enhance initiation of RNA transcription by an RNA polymerase. Oneexample of a promoter sequence includes the sequence SEQ ID NO. 535′-AATTTAATACGACTCACTATAGGGAGA-3′. Other examples of useful promotersequences are contained in various commercially available vectorsincluding, for example, pBluescript® vectors from Stratagene CloningSystems (San Diego, Calif.) or the pGEM™ vectors from Promega Corp.(Madison, Wis.)

In another aspect of the present invention the amplificationoligonucleotides bind to or cause elongation through sequencessubstantially corresponding to the following sequences:

SEQ ID NO: 23 GTCTTGAGAG GGAAAGCAGG GGAC

SEQ ID NO: 24 TATGTTACTC ACCCGTTCGC CACTCG(CC

SEQ ID NO: 19 CTGAAGAATA AGCACCGGCT AACTACGTGC AGCAGC

SEQ ID NO: 21 CGATGACGGT ACCTGAAGAA TAAGCACCGG CTAAC

SEQ ID NO: 20 AACGGCCTTT TCTTCCCTGA CAAAAGTCCT TTACAACCCG

SEQ ID NO: 22 GTCCTTTACA ACCCGAAGGC CTTC

SEQ ID NO: 47 GUCUUGAGAG GGAAAGCAGG GGAC

SEQ ID NO: 48 UAUGUUACUC ACCCGUUCGC CACUCGCC

SEQ ID NO: 49 CUGAAGAAUA AGCACCGGCU AACUACGUGC AGCAGC

SEQ ID NO: 50 CGAUGACGGU ACCUGAAGAA UAAGCACCGG CUAAC

SEQ ID NO: 51 AACGGCCUUU UCUUCCCUGA CAAAAGUCCU UUACAACCCG

SEQ ID NO: 52 GUCCUUUACA ACCCGAAGGC CUUC

Another aspect of the present invention includes kits that contain oneor more of the oligonucleotides of the present invention includingamplification oligonucleotides, helper oligonucleotides andhybridization assay probes. In preferred embodiments, a kit of thepresent invention includes at least one amplification oligonucleotideand one hybridization assay probe capable of distinguishing Neisseria,Neisseria meningitidis or Neisseria gonorrhoeae from othermicroorganisms.

Background descriptions of the use of nucleic acid hybridization todetect particular nucleic acid sequences are given in Kohne, U.S. Pat.No. 4,851,330 issued Jul. 25, 1989, and by Hogan et al., InternationalPatent Application No. PCT/US87/03009, entitled “Nucleic Acid Probes forDetection and/or Quantitation of Non-Viral Organisms”, both referenceshereby incorporated by reference herein. Hogan et al., supra, describemethods for determining the presence of a non-viral organism or a groupof non-viral organisms in a sample (e.g., sputum, urine, blood andtissue sections, food, soil and water).

In the most preferred embodiments, the compositions, probe mixes,probes, amplification primers, helper oligonucleotides and the like havea nucleotide sequence that consists of the specified nucleic acidsequence rather than substantially corresponding to the nucleic acidsequence. These most preferred embodiments use the sequence listed inthe sequence listing which forms part of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

The following terms have the indicated meanings in the specificationunless expressly indicated to have a different meaning.

By “target nucleic acid” is meant a nucleic acid having a targetnucleotide sequence.

By “oligonucleotide” is meant a single-stranded nucleotide polymer madeof more than 2 nucleotide subunits covalently joined together.Preferably between 10 and 100 nucleotide units are present, mostpreferably between 12 and 50 nucleotides units are joined together. Thesugar groups of the nucleotide subunits may be ribose, deoxyribose ormodified derivatives thereof such as 2′-O-methyl ribose. The nucleotidesubunits of an oligonucleotide may be joined by phosphodiester linkages,phosphorothioate linkages, methyl phosphonate linkages or by other rareor non-naturally-occurring linkages that do not prevent hybridization ofthe oligonucleotide. Furthermore, an oligonucleotide may have uncommonnucleotides or non-nucleotide moieties. An oligonucleotide as definedherein is a nucleic acid, preferably DNA, but may be RNA or have acombination of ribo- and deoxyribonucleotides covalently linked.Oligonucleotide probes and amplification oligonucleotides of a definedsequence may be produced by techniques known to those of ordinary skillin the art, such as by chemical or biochemical synthesis, and by invitro or in vivo expression from recombinant nucleic acid molecules,e.g., bacterial or retroviral vectors. As intended by this disclosure,an oligonucleotide does not consist of wild-type chromosomal DNA or thein vivo transcription products thereof. One use of a probe is as ahybridization assay probe; probes may also be used as in vivo or invitro therapeutic amplification oligomers or antisense agents to blockor inhibit gene transcription, or translation in diseased, infected, orpathogenic cells.

By “target nucleic acid sequence”, “target nucleotide sequence” or“target sequence” is meant a specific deoxyribonucleotide orribonucleotide sequence comprising all or a part of the nucleotidesequence of a single-stranded nucleic acid molecule, and thedeoxyribonucleotide or ribonucleotide sequence complementary thereto.

Nucleic acid hybridization is the process by which two nucleic acidstrands having completely or partially complementary nucleotidesequences come together under predetermined reaction conditions to forma stable, double-stranded hybrid with specific hydrogen bonds. Eithernucleic acid strand may be a deoxyribonucleic acid (DNA), a ribonucleicacid (RNA), or an analog of one of these nucleic acids; thushybridization can involve RNA:RNA hybrids, DNA:DNA hybrids, or RNA:DNAhybrids.

The term “hybridization” as used in this application, refers to theability of two completely or partly complementary single nucleic acidstrands to come together in an antiparallel orientation to form a stablestructure having a double-stranded region. The two constituent strandsof this double-stranded structure, sometimes called a hybrid, are heldtogether with hydrogen bonds. Although these hydrogen bonds mostcommonly form between nucleotides containing the bases adenine andthymine or uracil (A and T or U) or cytosine and guanine (C and G) onsingle nucleic acid strands, base pairing can form between bases who arenot members of these “canonical” pairs. Non-canonical base pairing iswell-known in the art. See e.g., The Biochemistry of the Nucleic Acids(Adams et al., eds., 1992).

“Stringent” hybridization assay conditions refer to conditions wherein aspecific hybridization assay probe is able to hybridize with targetnucleic acids (preferably rRNA or rDNA of a Neisseria, Neisseriameningitidis or Neisseria gonorrhoeae) over other nucleic acids presentin the test sample derived either from other microorganisms or fromhumans. It will be appreciated that these conditions may vary dependingupon factors including the GC content and length of the probe, thehybridization temperature, the composition of the hybridization reagentor solution, and the degree of hybridization specificity sought.Specific stringent hybridization conditions are provided in thedisclosure below.

As an example of specific stringent hybridization conditions useful indetecting Neisseria, Neisseria meningitidis or Neisseria gonorrhoeae,for the hybridization assay probes of this invention, a set of preferredstringent hybridization assay conditions was used. One preferred setcomprised hybridizing the target nucleic acid and hybridization probetogether in 100 μl of 0.05 M lithium succinate (pH 5.0), 6 M LiCl, 1%(w/v) lithium lauryl sulfate, (L.L.S.) 10 mM ethylene diaminetetraacetic acid (EDTA), 10 mM ethylene glycol bis (beta-amino ethylether) N,N,N′,N′ tetraacetic acid (EGTA) at 60° C. for 15 minutes, thenadding 300 μl of 0.15 M sodium tetraborate (pH 8.5), 1% (v/v) TRITON®X-100 at 60° C. for 5-7 minutes. Additional sets of stringenthybridization conditions can be determined after reading the presentdisclosure by those or ordinary skill in the art.

By “probe” is meant a single-stranded oligonucleotide having a sequencepartly or completely complementary to a nucleic acid sequence sought tobe detected, so as to hybridize thereto under stringent hybridizationconditions. The term “probe” is meant to exclude nucleic acids normallyexisting in nature. Purified oligonucleotide probes may be produced bytechniques known in the art such as chemical synthesis and by in vitroor in vivo expression from recombinant nucleic acid molecules, e.g.,retroviral vectors. Preferably probes are 10 to 100 nucleotides inlength. Probes may or may not have regions which are not complementaryto a target sequence, so long as such sequences do not substantiallyaffect hybridization under stringent hybridization conditions. If suchregions exist they may contain a 5′ promoter sequence and/or a bindingsite for RNA transcription, a restriction endonuclease recognition site,or may contain sequences which will confer a desired secondary ortertiary structure, such as a catalytic active site or a hairpinstructure on the probe, on the target nucleic acid, or both. A probe maybe labeled with a reporter group moiety such as a radioisotope, afluorescent or chemiluminescent moiety, with an enzyme or ligand, whichcan be used for detection or confirmation that the probe has hybridizedto the target sequence.

As used in this disclosure, the phrase “a probe (or oligonucleotide)having a nucleic acid sequence consisting essentially of a sequenceselected from” a group of specific sequences means that the probe, as abasic and novel characteristic, is capable of stably hybridizing to anucleic acid having the exact complement of one of the listed nucleicacid sequences of the group under stringent hybridization conditions. Anexact complement includes the corresponding DNA or RNA sequence.

The phrase “substantially corresponding to a nucleic acid sequence”means that the referred-to nucleic acid is sufficiently similar to thenucleic acid sequence such that the referred-to nucleic acid has similarhybridization properties to a nucleic acid sequence in that it wouldhybridize with the same target nucleic acid sequences under stringenthybridization conditions.

One skilled in the art will understand that substantially correspondingprobes and primers of the invention can vary from the referred-tosequence and still hybridize to the same target nucleic acid sequence.This variation from the nucleic acid may be stated in terms of apercentage of identical bases within the sequence or the percentage ofperfectly complementary bases between the probe or primer and its targetsequence. one skilled in the art will also understand that thisvariation could be expressed as the number of bases in a probe or primeror the number of mismatched bases of a probe that do not hybridize to acorresponding base of a target nucleic acid sequence. Probes or primersof the present invention substantially correspond to a nucleic acidsequence if these percentages are from 100% to 80% or from 0 basemismatches in a 10 nucleotide target sequence to 2 bases mismatched in a10 nucleotide target sequence.

In preferred embodiments, the percentage is from 100% to 85%. In morepreferred embodiments, this percentage is from 90% to 100%; in otherpreferred embodiments, this percentage is from 95% to 100%. One skilledin the art will understand the various modifications to thehybridization conditions that might be required at various percentagesof complementarity to allow hybridization to a specific target sequencewithout causing an unacceptable level of non-specific hybridization.

By “nucleic acid hybrid” or “hybrid” is meant a nucleic acid structurecontaining a double-stranded, hydrogen-bonded region, preferably ofbetween 10 and 100 nucleotides in length, most preferably of betweenabout 12 and 50 nucleotides in length, wherein each strand iscomplementary to the other and wherein the region is sufficiently stableunder stringent hybridization conditions to be detected by meansincluding but not limited to chemiluminescent or fluorescent lightdetection, autoradiography, or gel electrophoresis. Such hybrids maycomprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules or duplexmolecules containing analogs of these nucleic acids.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single strands to hybridize together in a stabledouble-stranded hydrogen-bonded region under stringent hybridizationconditions. When a contiguous sequence of nucleotides of onesingle-stranded region is able to form a series of “canonical”hydrogen-bonded base pairs with an analogous sequence of nucleotides ofthe other single-stranded region, such that A is paired with U or T andC is paired with G, the nucleotides sequences are “perfectly”complementary.

By “conservatively modified variants” is meant nucleic acids oroligonucleotides having a nucleotide sequence that is complementary to anucleic acid region of another nucleic acid, such region in turn beingperfectly complementary to a reference nucleic acid. Such conservativelymodified variants are able to stably hybridize to a target nucleic acidregion having a Neisseria, Neisseria meningitidis or Neisseriagonorrhoeae nucleotide sequence under stringent hybridizationconditions.

By “amplification oligonucleotide” is meant an oligonucleotide capableof hybridizing to a target nucleic acid sequence and acting as a primerfor nucleic acid synthesis or a promoter template (e.g., for synthesisof a complementary strand, thereby forming a functional promotersequence), or both, for the initiation of nucleic acid synthesis. If theamplification oligonucleotide is designed to initiate RNA synthesis, theoligonucleotide may contain nucleotide sequences which arenon-complementary to the target sequence, but are recognized by an RNApolymerase (such as T7, T3 and SP6 RNA polymerase). An amplificationoligonucleotide may or may not have a 3′ terminus which is modified toprevent or lessen the amount of primer extension. An amplificationoligonucleotide as defined herein will preferably be between 10 and 100nucleotides in length; most preferably between about 12 and 50nucleotides in length. While the amplification oligonucleotides of thepresent invention may be chemically synthesized or derived from avector, such oligonucleotides are not naturally-occurring nucleic acids.

By “nucleic acid amplification” or “target amplification” is meantincreasing the number of nucleic acid molecules having at least onetarget nucleic acid sequence.

By “antisense” or “negative sense” is meant having a nucleic sequencecomplementary to that of a reference nucleic acid sequence.

By “sense”, “same-sense” or “positive sense” is meant having a nucleicacid sequence analogous to that of a reference nucleic acid sequence.

By “helper oligonucleotide” is meant a nucleic acid probe designed tohybridize with the target nucleic acid at a different locus than that ofa labeled probe, thereby either increasing the rate of hybridization ofthe labeled probe, increasing the melting temperature (T_(m)) of thetarget:labeled probe hybrid, or both.

“Phylogenetically closely related” means that the organisms are closelyrelated to each other in an evolutionary sense and therefore would havea higher total nucleic acid sequence homology than organisms that aremore distantly related. Organisms occupying adjacent and next toadjacent positions on the phylogenetic tree are closely relatedorganisms occupying positions further away than adjacent or next toadjacent positions on the phylogenetic tree will still be closelyrelated if they have significant total nucleic acid sequence homology.

B. Hybridization Conditions and Probe/Primer Design

Hybridization reaction conditions, most importantly the temperature ofhybridization and the concentration of salt in the hybridizationsolution, can be selected to allow the amplification oligonucleotides orhybridization probes of the present invention to preferentiallyhybridize to nucleic acids having a target Neisseria nucleotidesequence, and not to other non-target nucleic acids suspected of beingpresent in the test sample. At decreased salt concentrations and/orincreased temperatures (called increased stringency) the extent ofnucleic acid hybridization decreases as hydrogen bonding between pairednucleotide bases in the double-stranded hybrid molecule is disrupted;this process is called “melting”.

Generally speaking, the most stable hybrids are those having the largestnumber of contiguous perfectly matched (i.e., hydrogen-bonded)nucleotide base pairs. Thus, such hybrids would usually be expected tobe the last to melt as the stringency of the hybridization conditionsincreases. However, a double-stranded nucleic acid region containing oneor more mismatched, “non-canonical”, or imperfect base pairs (resultingin weaker or non-existent base pairing at that position in thenucleotide sequence of a nucleic acid) may still be sufficiently stableunder conditions of relatively high stringency to allow the nucleic acidhybrid to be detected in a hybridization assay without cross reactingwith other, non-selected nucleic acids present in the test sample.

Hence, depending on the degree of similarity between the nucleotidesequences of the target nucleic acid and those of non-target nucleicacids belonging to phylogenetically distinct, but closely-relatedorganisms on one hand, and the degree of complementarity between thenucleotide sequences of a particular amplification oligonucleotide orhybridization probe and those of the target and non-target nucleic acidson the other, one or more mismatches will not necessarily defeat theability of the oligonucleotide to hybridize to that nucleic acid and notto non-target nucleic acids.

The hybridization assay probes of the present invention were chosen,selected, and/or designed to maximize the difference between the meltingtemperatures of the probe:target hybrid (T_(m), defined as thetemperature at which half of the potentially double-stranded moleculesin a given reaction mixture are in a single-stranded, denatured state)and the T_(m) of a mismatched hybrid formed between the probe and therRNA or rDNA of the phylogenetically most closely-related organismsexpected to be present in the test sample, but not sought to bedetected. While the unlabeled amplification oligonucleotides and helperoligonucleotides need not have such an extremely high degree ofspecificity as the labeled hybridization assay probe to be useful in thepresent invention, they are designed in a similar manner topreferentially hybridize to one or more target nucleic acids over othernucleic acids.

Probes specific for Neisseria meningitidis were designed using sequencesdetermined in prospective target areas using primers complementary tothe 16S rRNAs of strains of Neisseria including Neisseria gonorrhoeae(ATCC No. 19424), Neisseria meningitidis serogroup A (ATCC No. 13077),serogroup C (ATCC No. 23248) and serogroup L (ATCC No. 43828), clinicalisolates of Neisseria meinigitidis, Neisseria lactamica (ATCC NO.29193), Neisseria cinerea (ATCC NO. 14685), Neisseria mucosa (ATCC NO.19696), Neisseria sicca (ATCC NO. 29193) and Kingella kingae (ATCC No.23330). The nucleic acid sequence from phylogenetically near neighbors,including the published sequence of Neisseria gonorrhoeae NCTC 8375,Rossau et al. Nuc. Acids Res. 16:6227 were also used as comparisons withthe nucleic sequences from Neisseria meningitidis to determine variableregions.

To facilitate the identification of nucleic acid sequences to be used asprobes and amplification oligonucleotides, the nucleotide sequences fromdifferent species of organisms were first aligned to maximize homology.Within the rRNA molecule there is a close relationship between theoverall structure and function. This imposes restrictions onevolutionary changes in the primary sequence so that the secondarystructure is maintained. For example, if a base is changed on one sideof a helix, a compensating change may be evolutionarily made to theother side to preserve the complementarity (this is referred to asco-variance). This allows two very different sequences to be alignedusing the conserved primary sequence and also the conserved secondarystructure elements as points of reference. Potential target sequencesfor the hybridization probes were identified by noting variations in thehomology of the aligned sequences in certain discrete regions (variableregions) of the rRNA and rDNA sequences.

The sequence evolution at each of the variable regions is mostlydivergent. Because of the divergence, more distant phylogeneticrelatives of Neisseria meningitidis or Neisseria gonorrhoeae tend toshow greater variability in a given variable region thanphylogenetically closer relatives. The observed sufficient variationbetween Neisseria meningitidis and Neisseria gonorrhoeae species wasused to identify preferred target sites and design useful probes.

We have identified sequences which vary between Neisseria meningitidisand Neisseria gonorrhoeae, between these and other Neisseria species,and between members of the genus Neisseria and other organisms bycomparative analysis of rRNA sequences published in the literature ordetermined in the laboratory. Computers and computer programs which maybe used or adapted for the purposes herein disclosed are commerciallyavailable. We have seen sufficient variation between the targetorganisms and the closest phylogenetic relative likely to be found inthe same sample to design the present probes. The Neisseria meningitidisstrains have been classified into three sequence groups in the proberegion represented by serogroups A, C and L.

Merely identifying putatively unique potential target nucleotidesequences does not guarantee that a functionally species-specifichybridization assay probe may be made to hybridize to Neisseria rRNA orrDNA comprising that sequence. Various other factors will determine thesuitability of a nucleic acid locus as a target site forspecies-specific probes. Because the extent and specificity ofhybridization reactions such as those described herein are affected by anumber of factors, manipulation of one or more of those factors willdetermine the exact sensitivity and specificity of a particularoligonucleotide, whether perfectly complementary to its target or not.The importance and effect of various assay conditions are known to thoseskilled in the art as described in Hogan et al., PCT/US87/03009, andHogan and Hammond, U.S. Pat. No. 5,216,143, and Kohne, U.S. Pat. No.4,851,330 which share the same assignee as the present application andare hereby incorporated by reference herein.

The desired temperature of hybridization and the hybridization solutioncomposition (such as salt concentration, detergents and other solutes)can also greatly affect the stability of double-stranded hybrids.Conditions such as ionic strength and the temperature at which a probewill be allowed to hybridize to target must be taken into account inconstructing a group- or species-specific probe. The thermal stabilityof hybrid nucleic acids generally increases with the ionic strength ofthe reaction mixture. On the other hand, chemical reagents which disrupthydrogen bonds, such as formamide, urea, dimethyl sulfoxide andalcohols, can greatly reduce the thermal stability of the hybrids.

To maximize the specificity of a probe for its target, the subjectprobes of the present invention were designed to hybridize with theirtargets under conditions of high stringency. Under such conditions onlysingle nucleic acid strands having a high degree of complementarity willhybridize to each other; single nucleic acid strands without such a highdegree of complementarity will not form hybrids. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity which should exist between two nucleic acid strands inorder to form a hybrid. Stringency is chosen to maximize the differencein stability between the hybrid formed between the probe and the targetnucleic acid and potential hybrids between the probe and any non-targetnucleic acids present.

Proper specificity may be achieved by minimizing the length of the probehaving perfect complementarity to sequences of non-target organisms, byavoiding G and C rich regions of homology to non-target sequences, andby constructing the probe to contain as many destabilizing mismatches tonontarget sequences as possible. Whether a probe sequence is useful todetect only a specific type of organism depends largely on the thermalstability difference between probe:target hybrids versus potentialprobe:nontarget hybrids. In designing probes, the differences in theT_(m) values between these hybrids should be made as large as possible(preferably about 5° C. or more). Manipulation of the T_(m) can beaccomplished by changes to probe length and probe composition (GCcontent vs. AT content).

In general, the optimal hybridization temperature for oligonucleotideprobes of about 10-50 nucleotides in length is approximately 5° C. belowthe melting temperature for a given duplex. Incubation at temperaturesbelow the optimum temperature may allow mismatched base sequences tohybridize and can therefore decrease specificity. The longer the probe,the more hydrogen bonding between base pairs and, in general, the higherthe T_(m). Increasing the percentage of G and C also increases the T_(m)because G-C base pairs exhibit additional hydrogen bonding and thereforegreater thermal stability than A-T base pairs.

A preferred method to determine T_(m) measures hybridization using aHybridization Protection Assay (HPA) according to Arnold et al., U.S.Pat. No. 5,283,174 which enjoys exclusive ownership with the presentapplication and is incorporated by reference herein. T_(m) can bemeasured using HPA in -the following manner. A probe:target hybrid isformed in lithium succinate buffered solution (0.1 M lithium succinatebuffer, pH 5.0, 2 mM ethylenediamine tetraacetic acid (EDTA), 2 mMethylene glycol-bis (β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid(EGTA), 10% (w/v) lithium lauryl sulfate) using an excess amount oftarget. Aliquots of the hybrid are then diluted in the lithium succinatebuffered solution and incubated for five minutes at various temperaturesstarting below that of the anticipated T_(m) for example, 55° C. andincreasing in 2-5° C. increments. This solution is then diluted with amild alkaline borate buffer (0.15 M sodium tetraborate, pH. 7.6, 5%(v/v) TRITON® X-100) and incubated at a lower temperature (for example50° C.) for ten minutes. Under these conditions the acridinium esterattached to a single-stranded probe is hydrolyzed while the acridiniumester attached to hybridized probe is relatively protected fromhydrolysis. Thus, the amount of acridinium ester remaining isproportional to the amount of hybrid and can be measured by thechemiluminescence produced from the acridinium ester upon the additionof hydrogen peroxide followed by alkali. Chemiluminescence can bemeasured in a luminometer (e.g., Gen-Probe LEADER® I or LEADER® 50). Theresulting data are plotted as percent of maximum signal (usually fromthe lowest temperature) versus temperature. The T_(m) is defined as thetemperature at which 50% of the maximum signal remains. In addition tothe method above, T_(m) may be determined by isotopic methods well knownto those skilled in the art (e.g., Hogan et al., supra).

It should be noted that the T_(m) for a given hybrid varies depending onthe hybridization solution used. Factors such as the salt concentration,detergents, and other solutes can effect hybrid stability during thermaldenaturation (J. Sambrook, E. F. Fritsch and T. Maniatis, 2 MolecularCloning, ch. 11 (2d ed. 1989)). Conditions such as ionic strength andincubation temperature under which a probe will be used to hybridize totarget should be taken into account in constructing a probe. On theother hand, chemical reagents which disrupt hydrogen bonds such asformamide, urea, dimethylsulfoxide and alcohols, can greatly reduce thethermal stability of the hybrids.

To ensure the probe is specific for its target, it is desirable to haveprobes which hybridize only under conditions of high stringency. Underconditions of high stringency only highly complementary nucleic acidhybrids will form; hybrids without a sufficient degree ofcomplementarity will not form. Accordingly, the stringency of the assayconditions determines the amount of complementarity needed between twonucleic acid strands to form a hybrid. stringency is chosen to maximizethe difference in stability between the hybrid formed with the targetand other nucleic acid sequences.

The length of the target nucleic acid sequence and, accordingly, thelength of the probe sequence can also be important. In some cases, theremay be several sequences from a particular region, for example, avariable region varying in location and length, which yield probes withthe desired hybridization characteristics. In other cases, one probe maybe significantly better than another probe with a nucleotide sequencediffering by a single base. While it is possible for nucleic acids thatare not perfectly complementary to hybridize, the longest stretch ofperfectly homologous base sequence will generally determine hybridstability, with the composition of the base pairs also playing a role.

Regions of rRNA which form strong internal structures inhibitory tohybridization are less preferred target regions at least in assays inwhich helper probes are not used. Likewise, probe designs which resultin extensive self complementarity should be avoided. If one of the twostrands is wholly or partially involved in an intramolecular orintermolecular hybrid it will be less able to participate in theformation of a new intermolecular probe:target hybrid. Ribosomal RNAmolecules are known to form very stable intramolecular helices andsecondary structures by hydrogen bonding. By designing a hybridizationassay so that a substantial portion of the targeted sequence remains ina single-stranded state until hybridization with the probe, the rate andextent of hybridization between probe and target may be greatlyincreased. One way this may be accomplished is by choosing as a targetnucleotide sequence a sequence that is relatively uninvolved inintramolecular hydrogen-bonding. Alternatively or additionally, thehybridization assay probe may be used in a probe mix with helperoligonucleotides which can make the target site more accessible forhybridization with the hybridization assay probe.

A DNA target occurs naturally in a double-stranded form as does theproduct of the polymerase chain reaction (PCR). These double-strandedtargets are naturally inhibitory to hybridization with a probe andrequire denaturation prior to hybridization. Appropriate denaturationand hybridization conditions are known in the art (e.g., E. M. Southern,J. Mol. Bio. 98:503 (1975)).

A number of formulae are available which will provide an estimate of themelting temperature for perfectly matched oligonucleotides to theirtarget nucleic acids. One such formula,

T _(m)=81.5+16.6(log₁₀[Na⁺])+0.41(fraction G+C)−(600/N)

(where N=the length of the oligonucleotide in number of nucleotides)provides a good estimate for the T_(m) for oligonucleotides between 14and 60 or 70 nucleotides in length. From such calculations, subsequentempirical verification or “fine tuning” of the T_(m) may be made usingscreening techniques well known in the art. For further information onhybridization and oligonucleotide probes see, e.g., Sambrook et al., 2Molecular Cloning: A Laboratory Manual (Cold Springs Harbor LaboratoryPress 1989) hereby incorporated by reference herein (at Chapter 11).This reference, among others well known in the art, also providesestimates of the effect of mismatches on the T_(m) of a hybrid. Thus,from the known nucleotide sequence of a given region of the ribosomalRNA (or rDNA) of two or more organisms, oligonucleotides may be designedwhich will distinguish these organisms from one another.

C. Nucleic Acid Amplification

Preferably, the amplification oligonucleotides of the present inventionare oligodeoxynucleotides and are sufficiently long to be used as asubstrate for the synthesis of extension products by a nucleic acidpolymerase. Optimal primer length should take into account severalfactors, including the temperature of reaction, the structure and basecomposition of the primer, and how the primer is to be used. Forexample, for optimal specificity the oligonucleotide primer generallyshould contain at least about 12 nucleotides depending on the complexityof the target nucleic acid sequence. If such specificity is notessential, shorter primers may be used; in such a case, it may bedesirable to carry out reaction at cooler temperatures in order to formstable hybrid complexes with the template nucleic acid.

Useful guidelines for designing amplification oligonucleotides andprobes with desired characteristics are described herein. Our best modetarget regions contain at least two and preferably three conservedregions of Neisseria meningitidis or Neisseria gonorrhoeae nucleic acid.These regions are about 15-350 in length; preferably 15-150 nucleotidesin length.

The degree of amplification observed with a set of primers or promoterprimers depends on several factors, including the ability of theoligonucleotides to hybridize to their complementary sequences and theirability to be extended or copied enzymatically. While oligonucleotidesof different lengths and base composition may be used, oligonucleotidespreferred in this invention have target binding regions of 18-40 baseswith a predicted T_(m) to target of about 65° C.

Parameters which affect hybridization of a probe such as T_(m),complementarity and secondary structure of the target sequence alsoaffect primer hybridization and therefore performance. The degree ofnon-specific extension (primer-dimer or non-target copying) can alsoaffect amplification efficiency, therefore primers are selected to havelow self- or cross-complementarity, particularly at the 3′ ends of thesequence. Long homopolymer tracts and high GC content are avoided toreduce spurious primer extension. Computer programs are available to aidin this aspect of the design.

A nucleic acid polymerase used in conjunction with the amplificationoligonucleotides of the present invention refers to a chemical, physicalor biological agent which incorporates either ribo- ordeoxyribonucleotides, or both, into a nucleic acid polymer, or strand,in a template-dependent manner. Examples of nucleic acid polymerasesinclude DNA-directed DNA polymerases, RNA-directed DNA polymerases, andRNA-directed RNA polymerases. DNA polymerases bring about nucleic acidsynthesis in a template-dependent manner and in a 5′ to 3′ direction.Because of the antiparallel orientation of the two strands in adouble-stranded nucleic acid, this direction is from a 3′ region on thetemplate to a 5′ region on the template. Examples of DNA-directed DNApolymerases include E. coli DNA polymerase I, the thermostable DNApolymerase from Thermus aquaticus (Taq), and the large fragment of DNApolymerase I from Bacillus stearothermophilus (Bst). Examples of RNAdirected DNA polymerases include various retroviral reversetranscriptases, such as Moloney murine leukemia virus (MMLV) reversetranscriptase or avian myeloblastosis virus (AMV) reverse transcriptase.

During most nucleic acid amplification reactions, a nucleic acidpolymerase adds nucleotide residues to the 3′ end of the primer usingthe target nucleic acid as a template, thus synthesizing a secondnucleic acid strand having a nucleotide sequence partially or completelycomplementary to a region of the target nucleic acid. In many nucleicacid amplification reactions, the two strands comprising the resultingdouble-stranded structure must be separated by chemical or physicalmeans in order to allow the amplification reaction to proceed.Alternatively, the newly-synthesized template strand may be madeavailable for hybridization with a second primer or promoter-primer byother means—e.g. through strand displacement or the use of a nucleolyticenzyme which digests part or all of the original target strand. In thisway the process may be repeated through a number of cycles, resulting ina large increase in the number of nucleic acid molecules having thetarget nucleotide sequence.

Either the first or second amplification oligonucleotide, or both, maybe a promoter-primer. Such a promoter-primer usually contains nucleotidesequences that are not complementary to those of the target nucleic acidmolecule, or primer extension product(s). For example, Kacian and Fultz,U.S. Pat. No. 5,399,491 which is hereby incorporated by reference,describes various such oligonucleotides. These non-complementarysequences may be located 5′ to the complementary sequences on theamplification oligonucleotide, and may provide a locus for initiation ofRNA synthesis when made double-stranded through the action of a nucleicacid polymerase. The promoter thus provided may allow for the in vitrotranscription of multiple RNA copies of the target nucleic acidsequence. It will be appreciated that when reference is made to a primerin this specification, such reference is intended to include the primeraspect of a promoter-primer as well unless the context of the referenceclearly indicates otherwise.

In some amplification systems, for example the amplification method ofDattagupta et al., supra, the amplification oligonucleotides may contain5′ non-complementary nucleotides which assist in strand displacement.Furthermore, when used in conjunction with a nucleic acid polymerasehaving 5′ exonuclease activity, the amplification oligonucleotides mayhave modifications at their 5′ end to prevent enzymatic digestion.Alternatively, the nucleic acid polymerase may be modified to remove the5′ exonuclease activity, such as by treatment with a protease thatgenerates an active polymerase fragment with no such nuclease activity.In such a case the oligonucleotides need not be modified at their 5′end.

1. Preparation of Oligonucleotides

All of the amplification oligonucleotides of the present invention canbe readily prepared by methods known in the art. Preferably, the primersare synthesized using solid phase methods. For example, Caruthers, etal., describe using standard phosphoramidite solid phase chemistry tojoin nucleotides by phosphodiester linkages. Automated solid-phasechemical synthesis using cyanoethyl phosphoramidite precursors has beendescribed by Barone, et al., Nucleic Acids Research, 12:405 (1984).(Methods in Enzymology, Volume 143, pg. 287 (1987)). Likewise, Bhattdescribes a procedure for synthesizing oligonucleotides containingphosphorothioate linkages. (W092/04358, entitled “Method and Reagent forSulphurization of Organo-phosphorous Compounds”, which enjoys commonownership with the present invention.) Also, Klem et al., entitled“Improved Process for the Synthesis of oligomers”, PCT WO 92/07864,describe the synthesis of oligonucleotides having different linkagesincluding methylphosphonate linkages. The latter three references arehereby incorporated by reference herein. In addition, methods for theorganic synthesis of oligonucleotides are known to those of skill in theart, and are described in Sambrook, et al., supra, previouslyincorporated by reference herein.

Following synthesis and purification of a particular oligonucleotide,several different procedures may be utilized to purify and control thequality of the oligonucleotide. Suitable procedures includepolyacrylamide gel electrophoresis or high pressure liquidchromatography. Both of these procedures are well known to those skilledin the art.

All of the oligonucleotides of the present invention, whetherhybridization assay probes, amplification oligonucleotides, or helperoligonucleotides, may be modified with chemical groups to enhance theirperformance or to facilitate the characterization of amplificationproducts. For example, backbone-modified oligonucleotides such as thosehaving phosphorothioate or methylphosphonate groups which render theoligonucleotides resistant to the nucleolytic activity of certainpolymerases or to nuclease enzymes may allow the use of such enzymes inan amplification or other reaction. Another example of modificationinvolves using non-nucleotide linkers (e.g., Arnold, et al.,“Non-Nucleotide Linking Reagents for Nucleotide Probes”, EP 0 313 219hereby incorporated by reference herein) incorporated betweennucleotides in the nucleic acid chain which do not interfere withhybridization or the elongation of the primer. Amplificationoligonucleotides may also contain mixtures of the desired modified andnatural nucleotides.

The 3′ end of an amplification oligonucleotide may be blocked to preventinitiation of DNA synthesis as described by McDonough, et al., entitled“Nucleic Acid Sequence Amplification”, W094/03472 which enjoys commonownership with the present invention and is hereby incorporated byreference herein. A mixture of different 3′ blocked amplificationoligonucleotides, or of 3′ blocked and unblocked oligonucleotides mayincrease the efficiency of nucleic acid amplification, as describedtherein.

As disclosed above, the 5′ end of the oligonucleotides may be modifiedto be resistant to the 5′-exonuclease activity present in some nucleicacid polymerases. Such modifications can be carried out by adding anon-nucleotide group to the terminal 5′ nucleotide of the primer usingtechniques such as those described by Arnold, et al., supra, entitled“Non-Nucleotide Linking Reagents for Nucleotide Probes”, previouslyincorporated by reference herein.

Once synthesized, selected oligonucleotide probes may be labeled by anyof several well known methods (e, J. Sambrook, supra). Useful labelsinclude radioisotopes as well as non-radioactive reporting groups.Isotopic labels include ³H, ³⁵S, ³²p, ¹²⁵I, ⁵⁷Co and ¹⁴C. Isotopiclabels can be introduced into the oligonucleotide by techniques known inthe art such as nick translation, end labeling, second strand synthesis,the use of reverse transcription, and by chemical methods. When usingradiolabeled probes hybridization can be detected by autoradiography,scintillation counting, or gamma counting. The detection method selectedwill depend upon the particular radioisotope used for labeling.

Non-isotopic materials can also be used for labeling and may beintroduced internally into the nucleic acid sequence or at the end ofthe nucleic acid sequence. Modified nucleotides may be incorporatedenzymatically or chemically. Chemical modifications of the probe may beperformed during or after synthesis of the probe, for example, throughthe use of non-nucleotide linker groups as described by Arnold, et al.,supra “Non-Nucleotide Linking Reagents for Nucleotide Probes”,previously incorporated by reference herein. Non-isotopic labels includefluorescent molecules, chemiluminescent molecules, enzymes, cofactors,enzyme substrates, haptens or other ligands.

Preferably, the probes are labeled with an acridinium ester. Acridiniumester labeling may be performed as described by Arnold et al., U.S. Pat.No. 5,185,439, entitled “Acridinium Ester Labeling and Purification ofNucleotide Probes” issued Feb. 9, 1993 and hereby incorporated byreference herein.

2. Amplification of Neisseria rRNA and rDNA

The amplification oligonucleotides of the present invention are directedto particular Neisseria 16S rRNA nucleotide sequences, or their rDNAcounterparts. These amplification oligonucleotides may flank, overlap orbe contained within at least one of the target nucleotide sequences usedas a hybridization assay probe to detect the presence of Neisseria in anucleic acid amplification assay. The amplification oligonucleotidesdescribed and claimed herein comprise two sets of amplificationoligonucleotides. Members of the set of amplification oligonucleotidesare able to hybridize with a nucleic acid having or substantiallycorresponding to one of the following nucleotide sequences:

SEQ ID NO: 23 GTCTTGAGAG GGAAAGCAGG GGAC

SEQ ID NO: 24 TATGTTACTC ACCCGTTCGC CACTCGCC

SEQ ID NO: 19 CTGAAGAATA AGCACCGGCT AACTACGTGC AGCAGC

SEQ ID NO: 21 CGATGACGGT ACCTGAAGAA TAAGCACCGG CTAAC

SEQ ID NO: 20 AACGGCCTTT TCTTCCCTGA CAAAAGTCCT TTACAACCCG

SEQ ID NO: 22 GTCCTTTACA ACCCGAAGGC CTTC

SEQ ID NO: 47 GUCUUGAGAG GGAAAGCAGG GGAC

SEQ ID NO: 48 UAUGUUACUC ACCCGUUCGC CACUCGCC

SEQ ID NO: 49 CUGAAGAAUA AGCACCGGCU AACUACGUGC AGCAGC

SEQ ID NO: 50 CGAUGACGGU ACCUGAAGAA UAAGCACCGG CUAAC

SEQ ID NO: 51 AACGGCCUUU UCUUCCCUGA CAAAAGUCCU UUACAACCCG and

SEQ ID NO: 52 GUCCUUUACA ACCCGAAGGC CUUC

In preferred embodiments, these amplification oligonucleotides have orsubstantially correspond to the following sequences:

SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC

SEQ ID NO: 6 GGCGAGTGGC. GAACGGGTGA GTAACATA

SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG

SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG

SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT

SEQ ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC

SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC

SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA

SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG

SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG

SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU

SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC

These oligonucleotides may also have additional, non-complementary basesat their 5′ end comprising a promoter sequence able to bind an RNApolymerase and direct RNA transcription using the target nucleic acid asa template. For example the promoter, SEQ ID NO: 53 AATTTAATACGACTCACTAT AGGGAGA may be used.

All of the amplification oligonucleotides of the present invention mayhave sequences which do not contain modifications or additions to thesesequences. The amplification oligonucleotides may also or alternativelyhave modifications, such as blocked 3′ and/or 5′ termini or additionsincluding but not limited to the addition of a specific nucleotidesequence that is recognized by an RNA polymerase, (e.g., the promotersequence for T7, T3, or SP6 RNA polymerase), addition of sequences whichenhance initiation or elongation of RNA transcription by an RNApolymerase, or sequences which may provide for intramolecular basepairing and encourage the formation of secondary or tertiary nucleicacid structures.

Amplification oligonucleotides are used in a nucleic acid amplificationprocedure, such as the polymerase chain reaction or an amplificationreaction using RNA polymerase, DNA polymerase and RNAse H or itsequivalent, as described by Kacian and Fultz supra, Dattagupta et al.,supra, and by Sninsky et al., U.S. Pat. No. 5,079,351; all herebyincorporated by reference herein, the first two of which enjoy commonownership with the present invention.

A wide variety of methods are available to detect an amplified targetsequence. For example, the nucleotide substrates or the primers caninclude a detectable label which is incorporated into newly synthesizedDNA. The resulting labeled amplification product is then separated fromthe unused labeled nucleotides or primers and the label is detected inthe separated product fraction.

Substances which can serve as useful detectable labels are well known inthe art and include radioactive isotopes, fluorescent compounds,chemiluminescent compounds, chromophores, as well as ligands such asbiotin and haptens which, while not directly detectable, can be readilydetected by a reaction with labeled forms of their specific bindingpartners, e.g., avidin and antibodies, respectively.

Another approach is to detect the amplification product by hybridizationwith a detectably labeled nucleic acid probe and measuring the resultinghybrids in any conventional manner. In particular, the product can beassayed by hybridizing a chemiluminescent acridinium ester-labelednucleic acid probe to the target sequence, selectively hydrolyzing theacridinium ester present on unhybridized probe, and measuring thechemiluminescence produced from the remaining acridinium ester in aluminometer. (see, e.g., Arnold, et al., supra, U.S. Pat. No. 5,283,174,and Nelson, et al., “Non-Isotopic DNA Probe Technologies”, AcademicPress, San Diego (Kricka, ed. 1992) both references hereby incorporatedby reference herein.)

D. Oligonucleotide Hybridization Assay Probes to Neisseria Meningitidisor Neisseria Gonorrhoeae rRNA and rDNA

The oligonucleotide hybridization assay probes disclosed and claimedherein are able to preferentially hybridize to target nucleic acids ofNeisseria meningitidis rRNA or rDNA nucleotide sequences over nucleicacids of phylogenetically closely related bacterial species. Thesehybridization assay probes were designed, selected and/or chosen basedupon a comparison of the nucleotide sequences of corresponding. regionsof the ribosomal RNA of Neisseria meningitidis and said phylogeneticallyclosely-related species. In preferred embodiments these probesselectively hybridize to the nucleic acids of Neisseria meningitidisover the nucleic acids of Neisseria gonorrhoeae.

The present invention contemplates oligonucleotide hybridization probesthat selectively hybridize to the nucleic acids of Neisseriameningitidis and not to the nucleic acids of Neisseria gonnorhoeae andinclude Neisseria menigitidis nucleic acid sequences having orsubstantially corresponding to the following nucleic acid sequences:

SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG

SEQ ID NO: 12 GGCTGTTGCT AATACCAGCG

SEQ ID NO: 15 CGCTGATATT AGCAACAGCC

SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC

SEQ ID NO: 25 GGCUGUUGCU AAUAUCAGCG

SEQ ID NO: 26 GGCUGUUGCU AAUACCAGCG

SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC

SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC

A number of oligonucleotide hybridization assay probes of the presentinvention preferably hybridize to target nucleic acids containingNeisseria gonorrhoeae rRNA or rDNA nucleotide sequences over nucleicacids of other phylogenetically closely related bacterial species. Inpreferred embodiments, these hybridization assay probes can distinguishNeisseria gonorrhoeae nucleic acids from Neisseria meningitidis.

The hybridization probes of the present invention that selectivelyhybridize to nucleic acids derived from Neisseria gonorrhoeae and not tothe nucleic acids of Neisseria meningitidis have or substantiallycorrespond to the following nucleotide sequences:

SEQ ID NO 1: GAACGTACCG GGTAGCGG

SEQ ID NO 3: GCCAATATCG GCGGCCGATG

SEQ ID NO 29: CCGCTACCCG GTACGTTC

SEQ ID NO 30: CATCGGCCGC CGATATTGGC

SEQ ID NO 31: GAACGUACCG GGUAGCGG

SEQ ID NO 32: GCCAAUAUCG GCGGCCGAUG

SEQ ID NO 33: CCGCUACCCG GUACGUUC

SEQ ID NO 34: CAUCGGCCGC CGAUAUUGGC

The oligonucleotide hybridization assay probes of the present inventionare preferably labeled with a reporter group moiety such as aradioisotope, a fluorescent or chemiluminescent moiety, with an enzymeor other ligand, which can be used for detection or confirmation thatthe probe has hybridized to the target sequence. The Applicant mostprefers the use of chemiluminescent acridinium esters as labels. Seee.g. Arnold et al., U.S. Pat. No. 5,185,439, previously incorporated byreference herein. The assay probe is mixed with a sample suspected ofcontaining a nucleic acid having the target sequence under hybridizationconditions suitable for allowing annealing of the two strands byhydrogen bonding in the region of complementarity.

The probe may also be combined with one or more unlabeled helperoligonucleotides to facilitate binding to the nucleic acid having thetarget Neisseria meningitidis or Neisseria gonorrhoeae nucleotidesequence. The probe then hybridizes to the target nucleic acid presentin the sample; the resulting hybrid duplex may be separated. anddetected by various techniques well known in the art, such ashydroxyapatite adsorption and radioactive monitoring. Also includedamong these techniques are those that involve selectively degrading thelabel present on unhybridized probe and then measuring the amount oflabel associated with the remaining hybridized probe, as disclosed inArnold et al., U.S. Pat. No. 5,283,174, which enjoys common ownershipwith the present application and is incorporated by reference herein.This latter technique is presently preferred by the Applicants.

E. Helper Oligonucleotides Used in the Detection of Neisseria

Specific helper oligonucleotides were used to facilitate thehybridization of the hybridization assay probes to the target nucleicacid. Helper oligonucleotides are described in Hogan and Milliman, U.S.Pat. No. 5,030,557 entitled Means and Method for Enhancing Nucleic AcidHybridization, which enjoys common ownership with the presentapplication and is hereby incorporated by reference herein.

Helper probes are selected to hybridize to nucleic acid sequenceslocated near the region targeted by the hybridization assay probe.Hybridization of the helper probe alters the secondary and tertiarystructure of the target nucleic acid, facilitating the hybridization ofthe probe to the target nucleic acid.

Specific helper oligonucleotides for facilitating the specific detectionof Neisseria meningitidis nucleic acids have or substantially correspondto one of these nucleotide sequences:

SEQ ID NO: 13 GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA

SEQ ID NO: 14 GCTGATGACG GTACCTGAAG AATAAGCACC GGC

SEQ ID NO: 17 TTTTCTTCCC TGACAAAAGT CCTTTACAAC CCGAAGGC

SEQ ID NO: 18 GCCGGTGCTT ATTCTTCAGG TACCGTCATC AGC

SEQ ID NO: 35 GCCUUCGGGU UGUAAAGGAC UUUUGUCAGG GAAGAAAA

SEQ ID NO: 36 GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC

SEQ ID NO: 37 UUUUCUUCCC UGACAAAAGU CCUUUACAAC CCGAAGGC

SEQ ID NO: 38 GCCGGUGCUU AUUCUUCAGG UACCGUCAUC AGC

In preferred embodiments, hybridization probes directed to Neisseriameningitidis nucleic acids substantially correspond to SEQ ID NOS: 11,12, 25 or 26 used in a probe mixture together with a helperoligonucleotide having or substantially corresponding to the nucleotidesequence of:

SEQ ID NOS: 13, 14, 35 and 36

In other embodiments, a hybridization assay probe directed to Neisseriameningitidis nucleic acids substantially corresponding to

SEQ ID NOS: 15, 16, 27 or 28

is used in a probe mixture together with a helper oligonucleotidehaving. or substantially corresponding to a nucleotide sequence of:

SEQ ID NOS: 17, 18, 37 and 38

In a preferred embodiment, a hybridization probe directed to Neisseriagonorrhoeae ribosomal nucleic acid substantially corresponding to

SEQ ID NOS: 1 or 31

is used in a mixture together with a helper oligonucleotide having orsubstantially corresponding to the nucleotide sequence of:

SEQ ID NOS: 2 or 39

In other preferred embodiments, a hybridization probe directed toNeisseria gonorrhoeae nucleic acids substantially corresponding to

SEQ ID NOS: 3 or 32

is used in a probe mixture together with a helper oligonucleotide havingor substantially corresponding to a nucleotide sequence of:

SEQ ID NOS: 4 or 40

Helper oligonucleotides generally may be used under stringenthybridization conditions, but are not necessarily species specific.

F. Nucleic Acid Compositions

In another related aspect, the invention features compositionscomprising a nucleic acid hybrid between a hybridization assay probe anda nucleic acid sequence substantially complementary thereto(probe:target). One use of the hybrid formed between probe and target isto detect the presence of a target sequence. For example, acridiniumester (“AE”) present in hybrids is resistant to hydrolysis in alkalisolution whereas AE present in single-stranded nucleic acid ishydrolyzed in alkali solution (Arnold et al., entitled “HomogenousProtection Assay,” EPO application number 88308767.8, publication number309230, and by U.S. Pat. No. 5,238,174 hereby incorporated byreference). Thus, presence of target nucleic acids can be detected,after hydrolysis of the unbound AE-labeled probe, by measuringchemiluminescence of acridinium ester remaining associated with thenucleic acid hybrid.

The present invention also. contemplates compositions comprising anucleic acid hybrid between an amplification oligonucleotide and anucleic acid sequence substantially complementary thereto.(primer:target). One use the nucleic acid hybrid formed between primerand target is to provide an initiation site for a nucleic acidpolymerase at the 3′ end of the amplification oligonucleotide. Forexample, hybrids may form an initiation site for reverse transcriptase,DNA polymerases such as Taq polymerase or T4 DNA polymerase and RNApolymerases such as, T7 polymerase, SP6 polymerase, T3 polymerases andthe like.

The present invention also features compositions comprising nucleic acidhybrids between a helper oligonucleotide and a nucleic acid sequencesubstantially complementary thereto (helper oligonucleotide:target). Oneuse of the hybrid between the helper oligonucleotide and target is tomake available a particular nucleic acid sequence for hybridization. Forexample, a hybrid between a helper oligonucleotide and its target maymake a nucleic acid sequence capable of hybridizing to the targetsequence available for hybridization with a hybridization probe. A fulldescription of the. use of helper oligonucleotides is provided in Hoganand Milliman, U.S. Pat. No. 5,030,557.

Compositions of the present invention include compositions for detectingNeisseria meningitidis nucleic acid comprising a nucleic acid hybridformed between a nucleic acid derived from Neisseria meningitidis and anoligonucleotide having a nucleic acid sequence substantiallycorresponding to at least one of the nucleic acid sequences thatfollows:

SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG

SEQ ID NO: 12 GGCTGTTGCT AATACCAGCG

SEQ ID NO: 15 CGCTGATATT AGCAACAGCC

SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC

SEQ ID NO: 25 GGCUGUUGCU AAUAUCAGCG

SEQ ID NO: 26 GGCUGUUGCU AAUACCAGCG

SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC

SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC

SEQ ID NO: 13 GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA

SEQ ID NO: 14 GCTGATGACG GTACCTGAAG AATAAGCACC GGC

SEQ ID NO: 17 TTTTCTTCCC TGACAAAAGT CCTTTACAAC CCGAAGGC

SEQ ID NO: 18 GCCGGTGCTT ATTCTTCAGG TACCGTCATC AGC

SEQ ID NO: 35 GCCUUCGGGU UGUAAAGGAC UUUGUCAGG GAAGAAAA

SEQ ID NO: 36 GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC

SEQ ID NO: 37 UUUUCUUCCC UGACAAAAGU CCUUUACAAC CCGAAGGC

SEQ ID NO: 38 GCCGGUGCUU AUUCUUCAGG UACCGUCAUC AGC

SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC

SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA GTAACATA

SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG

SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG

SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT

SEQ ID NO: 10GAAGGCCTTC GGGTTGTAAA GGAC

SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC

SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA

SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG

SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG

SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU

SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC

Preferred compositions of the present invention include compositions fordetecting Neisseria meningitidis comprising a nucleic acid hybrid formedbetween a nucleic acid derived from Neisseria meningitidis and anoligonucleotide having a nucleic acid sequence substantiallycorresponding to at least one of the nucleic acid sequences thatfollows:

SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG

SEQ ID NO: 12 GGCTGTTGCT AATACCAGCG

SEQ ID NO: 15 CGCTGATATT AGCAACAGCC

SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC

SEQ ID NO: 25 GGCUGUUGCU AAUAUCAGCG

SEQ ID NO: 26 GGCUGUUGCU AAUACCAGCG

SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC

SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid hybrid formed between aNeisseria meningitidis-derived nucleic acid and a hybridization assayprobe having a nucleic acid sequence substantially corresponding to:

SEQ ID NO: 11 or SEQ ID NO: 25;

and which may also optionally contain a helper oligonucleotidehybridized to said nucleic acid which has a nucleic acid sequence whichsubstantially corresponds to one of the following nucleic acidsequences:

SEQ ID NOS: 13, 14, 35 or 36.

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid hybrid formed between aNeisseria meningitidis-derived nucleic acid and a hybridization assayprobe having a nucleic acid sequence substantially corresponding to:

SEQ ID NO: 12 or SEQ ID NO: 26;

and which may also optionally contain a helper oligonucleotidehybridized to said nucleic acid which has a nucleic acid sequence whichsubstantially corresponds to one of the following nucleic acidsequences:

SEQ ID NOS: 13, 14, 35 or 36.

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid hybrid formed between aNeisseria meningitidis-derived nucleic acid and a hybridization assayprobe having a nucleic acid sequence substantially corresponding to:

SEQ ID NO: 15 or SEQ ID NO: 27;

and which may also optionally contain a helper oligonucleotidehybridized to said nucleic acid which has a nucleic acid sequence whichsubstantially corresponds to one of the following nucleic acidsequences:

SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid hybrid formed between aNeisseria meningitidis-derived nucleic acid and a hybridization assayprobe having a nucleic acid sequence substantially corresponding to:

SEQ ID NO: 16or SEQ ID NO: 28;

and which may also optionally contain a helper oligonucleotidehybridized to said nucleic acid which has a nucleic acid sequence whichsubstantially corresponds to one of the following nucleic acidsequences:

SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detectingNeisseria gonorrhoeae having a nucleic acid hybrid formed between aNeisseria gonorrhoeae-derived nucleic acid and a hybridization assayprobe having a nucleic acid sequence substantially corresponding to:

SEQ ID NO: 1 or SEQ ID NO: 31;

and which may also optionally contain a helper oligonucleotidehybridized to said nucleic acid which has a nucleic acid sequence whichsubstantially corresponds to one of the following nucleic acidsequences:

SEQ ID NOS: 2 or 39.

The present invention also contemplates compositions for detectingNeisseria gonorrhoeae having a nucleic acid hybrid formed between aNeisseria gonorrhoeae-derived nucleic acid and a hybridization assayprobe having a nucleic acid sequence substantially corresponding to:

SEQ ID NO: 3 or SEQ ID NO: 32;

and which may also optionally contain a helper oligonucleotidehybridized to said nucleic acid which has a nucleic acid sequence whichsubstantially corresponds to one of the following nucleic acidsequences:

SEQ ID NOS: 4 or 40.

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid derived from Neisseriameningitidis and an oligonucleotide having a nucleic acid sequencesubstantially corresponding to

SEQ ID NOS: 7 or 43

and/or an oligonucleotide having nucleic acid sequence substantiallycorresponding to at least one nucleic acid sequence that follows:

SEQ ID NOS: 9 or 45

and optionally has a hybridization assay probe capable of hybridizing toa Neisseria meningitidis nucleic acid and which has a nucleic acidsequence substantially corresponding to one of the following nucleicacid sequences:

SEQ ID NOS: 11, 15, 25 or 27

and which may also optionally contain a helper oligonucleotide which hasa nucleic acid sequence which substantially corresponds to one of thefollowing nucleic acid sequences:

SEQ ID NOS: 13, 14, 35 or 36.

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid derived from Neisseriameningitidis and an oligonucleotide with a nucleic acid sequencesubstantially corresponding to

SEQ ID NOS: 7 or 43

and/or which also has an oligonucleotide having nucleic acid sequencesubstantially corresponding to at least one nucleic acid sequence thatfollows:

SEQ ID NOS: 9 or 45

and optionally has a hybridization assay probe capable of hybridizing toNeisseria meningitidis nucleic acids and which has a nucleic acidsequence substantially corresponding to one of the following nucleicacid sequences:

SEQ ID NOS: 12, 16, 26 or 28

and which may also optionally contain a helper oligonucleotide which hasa nucleic acid sequence which substantially corresponds to one of thefollowing nucleic acid sequences:

SEQ ID NOS: 13, 14, 35 or 36.

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid derived from Neisseriameningitidis and an oligonucleotide with a nucleic acid sequencesubstantially corresponding to

SEQ ID NOS: 7 or 43

and/or which also has an oligonucleotide having nucleic acid sequencesubstantially corresponding to at least one nucleic acid sequence thatfollows:

SEQ ID NOS: 9 or 45

and optionally has a hybridization assay probe capable of hybridizing toa Neisseria meningitidis nucleic acid which has a nucleic acid sequencesubstantially corresponding to one of the following nucleic acidsequences:

SEQ ID NOS: 15, 11, 27 or 25

and which may also optionally contain a helper oligonucleotide which hasa nucleic acid sequence which substantially corresponds to one of thefollowing nucleic acid sequences:

SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid derived from Neisseriameningitidis and an oligonucleotide with a nucleic acid sequencesubstantially corresponding to

SEQ ID NOS: 7 or 43

and/or which also has an oligonucleotide having nucleic acid sequencesubstantially corresponding to at least one nucleic acid sequence thatfollows:

SEQ ID NOS: 9 or 45

and optionally has a hybridization assay probe capable of hybridizing toa Neisseria meningitidis nucleic acid which has a nucleic acid sequencesubstantially corresponding to one of the following nucleic acidsequences:

SEQ ID NOS: 16, 12, 28 or 20

and which may also optionally contain a helper, oligonucleotide whichhas a nucleic acid sequence which substantially corresponds to one ofthe following nucleic acid sequences:

SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid derived from Neisseriameningitidis and an oligonucleotide with a nucleic acid sequencesubstantially corresponding to

SEQ ID NOS: 8 or 44

and/or which also has an oligonucleotide having nucleic acid sequencesubstantially corresponding to at least one nucleic acid sequence thatfollows:

SEQ ID NOS: 10 or 46.

and optionally has a hybridization assay probe capable of hybridizing toa Neisseria meningitidis nucleic acid which has a nucleic acid sequencesubstantially corresponding to one of the following nucleic acidsequences:

SEQ ID NOS: 15, 11, 27 or 25

and which may also optionally contain a helper oligonucleotide which hasa nucleic acid sequence which substantially corresponds to one of thefollowing nucleic acid sequences:

SEQ ID NOS: 17, 18, 37 or 38.

The present invention also contemplates compositions for detectingNeisseria meningitidis having a nucleic acid derived from Neisseriameningitidis and an oligonucleotide with a nucleic acid sequencesubstantially corresponding to

SEQ ID NOS: 8 or 44

and/or which also has an oligonucleotide having nucleic acid sequencesubstantially corresponding to at least one nucleic acid sequence thatfollows:

SEQ ID NOS: 10 or 46

and optionally has a hybridization assay probe capable of hybridizing toa Neisseria meningitidis nucleic acid which has a nucleic acid sequencesubstantially corresponding to one of the following nucleic acidsequences:

SEQ ID NOS: 16, 12, 28 or 26

and which may also optionally contain a helper oligonucleotide which hasa nucleic acid sequence which substantially corresponds to one of thefollowing nucleic acid sequences:

SEQ ID NOS: 17, 18, 37 or 38.

Preferred compositions of the present invention include compositions fordetecting Neisseria gonorrhoeae comprising a nucleic acid hybrid formedbetween a nucleic acid derived from Neisseria gonorrhoeae and anoligonucleotide having a nucleic acid sequence substantiallycorresponding to at least one of the nucleic acid sequences that follow:

SEQ ID NO 1: GAAGCTACCG GGTAGCGG

SEQ ID NO 3: GCCAATATCG GCGGCCGATG

SEQ ID NO 29: CCGCTACCCG GTACGTTC

SEQ ID NO 30: CATCGGCCGC CGATATTGGC

SEQ ID NO 31: GAACGUACCG GGUAGCGG

SEQ ID NO 32: GCCAAUAUCG GCGGCCGAUG

SEQ ID NO 33: CCGCUACCCG GUACGUUC

SEQ ID NO 34: CAUCGGCCGC CGAUAUUGGC

SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC

SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA GTAACATA

SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG

SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG

SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT

SEQ ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC

SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC

SEQ ID NO: 42 GGCGAGUGGC GAACGGGUGA GUAACAUA

SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG

SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG

SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU

SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC

SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG

SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC TACGTG

SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG

SEQ ID NO: 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG

More preferred compositions of the present invention includecompositions for detecting Neisseria gonorrhoeae comprising a nucleicacid hybrid formed between a nucleic acid derived from Neisseriagonorrhoeae and an oligonucleotide having a nucleic acid sequencesubstantially corresponding to at least one of the nucleic acidsequences that follow:

SEQ ID NO 1: GAACGTACCG GGTAGCGG

SEQ ID NO 3: GCCAATATCG GCGGCCGATG

SEQ ID NO 31: GAACGUACCG GGUAGCGG

SEQ ID NO 32: GCCAAUAUCG GCGGCCGAUG

SEQ ID NO 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG

SEQ ID NO 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC TACGTG

SEQ ID NO 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG

SEQ ID NO 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG

The present invention also contemplates compositions for detectingNeisseria gonorrhoeae having a nucleic acid derived from Neisseriagonorrhoeae and an oligonucleotide with a nucleic acid sequencesubstantially corresponding to

SEQ ID NOS: 5 or 41

and/or which also optionally has an oligonucleotide having nucleic acidsequence substantially corresponding to at least one nucleic acidsequence that follows:

SEQ ID NOS: 6 or 42

and optionally has a hybridization assay probe capable of hybridizing toa Neisseria gonnorhoeae nucleic acid and which has a nucleic acidsequence substantially corresponding to one of the following nucleicacid sequences:

SEQ ID NOS: 1, 29, 31 or 33

and which may also optionally contain a helper oligonucleotide which asa nucleic acid sequence which substantially corresponds to one of thefollowing nucleic acid sequences:

SEQ ID NOS: 2 or 39

The present invention also contemplates compositions for detectingNeisseria gonnorhoeae having a nucleic acid derived from Neisseriagonnorhoeae and an oligonucleotide with a nucleic acid sequencesubstantially corresponding to

SEQ ID NOS: 7 or 42

and/or which optionally has an oligonucleotide nucleic acid sequencesubstantially corresponding to at least one nucleic acid sequence thatfollows:

SEQ ID NOS: 9 or 45

and which optionally has a hybridization assay probe capable ofhybridizing to a Neisseria gonnorhoeae nucleic acid and which has anucleic acid sequence substantially corresponding to one of thefollowing nucleic acid sequences:

SEQ ID NOS: 3, 30, 32 or 34

and which may also optionally contain a helper oligonucleotide which hasa nucleic acid sequence which substantially corresponds to one of thefollowing nucleic acid sequences:

SEQ ID NOS: 4 or 40

and which also optionally has an oligonucleotide having a nucleic acidsequence substantially corresponding to at least one nucleic acidsequence that follows:

SEQ ID NOS: 8 or 44

The present invention also contemplates compositions for detectingNeisseria gonnorhoeae having a nucleic acid derived from Neisseriagonnorhoeae and an oligonucleotide with a nucleic acid sequencesubstantially corresponding to

SEQ ID NOS: 10 or 46

and optionally has a hybridization assay probe capable of hybridizing toNeisseria gonnorhoeae nucleic acids and which has a nucleic acidsequence substantially corresponding to one of the following nucleicacid sequences:

SEQ ID NOS: 3, 30, 32 or 34

and/or which may also optionally contain a helper oligonucleotide whichhas a nucleic acid sequence which substantially corresponds to one ofthe following nucleic acid sequences:

SEQ ID NOS: 4 or 40

The present invention also contemplates nucleic acid hybrids comprisingprobes of the present invention and also at least one helperoligonucleotide that has a nucleic acid sequence substantiallycorresponding to at least one of the nucleic acid sequences thatfollows:

SEQ ID NO: 13 GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA

SEQ ID NO: 14 GCTGATGACG GTACCTGAAG AATAAGCACC GGC

SEQ ID NO: 17 TTTTCTTCCC TGACAAAAGT CCTTTACAAC CCGAAGGC

SEQ ID NO: 18 GCCGGTGCTT ATTCTTCAGG TACCGTCATC AGC

SEQ ID NO: 35 GCCUUCGGGU UGUAAAGGAC UUUUGUCAGG GAAGAAAA

SEQ ID NO: 36 GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC

SEQ ID NO: 37 UUUUCUUCCC UGACAAAAGU CCUUUACAAC CCGAAGGC

SEQ ID NO: 38 GCCGGUGCUU AUUCUUCAGG UACCGUCAUC AGC

SEQ ID NO: 2 GGGATAACTG ATCGAAAGAT CAGCTAATAC CGCATACG

SEQ ID NO: 4 ACGGTACCTG AAGAATAAGC ACCGGCTAAC TACGTG

SEQ ID NO: 39 GGGAUAACUG AUCGAAAGAU CAGCUAAUAC CGCAUACG

SEQ ID NO: 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG

The present invention also contemplates compositions for amplifyingNeisseria nucleic acids comprising a nucleic acid hybrid formed betweena Neisseria nucleic acid and an oligonucleotide having a nucleic acidsequence substantially corresponding to a nucleic acid sequence selectedfrom the group consisting of:

SEQ ID NO: 5 GTCCCCTGCT TTCCCTCTCA AGAC

SEQ ID NO: 6 GGCGAGTGGC GAACGGGTGA GTAACATA

SEQ ID NO: 41 GUCCCCUGCU UUCCCUCUCA AGAC

SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG

SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG

SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG

SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG

SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT

SEQ ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC

SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU

SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC

G. Assay Methods

The present invention contemplates various methods for assaying for thepresence of Neisseria meningitidis or Neisseria gonorrhoeae nucleic acidwithin a sample. One skilled in the art will understand that the exactassay conditions, probes or primers used will vary depending on theparticular assay format used and the source of the sample.

Generally, the present invention contemplates methods for detecting thepresence of Neisseria meningitidis by contacting a test sample understringent hybridization conditions with a nucleic acid hybridizationassay probe capable of preferentially hybridizing under stringenthybridization assay conditions to a Neisseria meningitidis targetnucleic acid over nucleic acids from Neisseria gonorrhoeae, said targetnucleic acid having a nucleic acid sequence substantially correspondingto a sequence selected from the group consisting of:

SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG

SEQ ID NO: 12 GGCTGTTGCT AATACCAGCG

SEQ ID NO: 15 CGCTGATATT AGCAACAGCC

SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC

SEQ ID NO: 25 GGCUGUUGCU AAUAUCAGCG

SEQ ID NO: 26 GGCUGUUGCU AAUACCAGCG

SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC

SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC

Preferred methods for detecting the presence of Neisseria meningitidisinclude the step of contacting a test sample under stringenthybridization conditions with a nucleic acid hybridization assay probecapable of preferentially hybridizing under stringent hybridizationassay conditions to a Neisseria meningitidis target nucleic acidsequence over nucleic acid sequences of Neisseria gonorrhoeae, saidtarget nucleic acid sequence substantially corresponding to a sequenceselected from the group consisting of:

SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG

SEQ ID NO: 12 GGCTGTTGCT AATACCAGCG

SEQ ID NO: 15 CGCTGATATT AGCAACAGCC

SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC

Preferred methods for detecting the presence of Neisseria gonorrhoeaeinclude the step of contacting a test sample under stringenthybridization conditions with a nucleic acid hybridization assay probecapable of preferentially hybridizing under stringent hybridizationassay conditions to aNeisseria gonorrhoeae target nucleic acid sequenceover a nucleic acid sequence of Neisseria meningitidis, said targetnucleic acid sequence substantially corresponding to a sequence selectedfrom the group consisting of:

SEQ ID NO 1: GAACGTACCG GGTAGCGG

SEQ ID NO 3: GCCAATATCG GCGGCCGATG

SEQ ID NO 29: CCGCTACCCG GTACGTTC

SEQ ID NO 30: CATCGGCCGC CGATATTGGC

SEQ ID NO 31: GAACGUACCG GGUAGCGG

SEQ ID NO 32: GCCAAUAUCG GCGGCCAUG

SEQ ID NO 33: CCGCUACCCG GUACGUUC

SEQ ID NO 34: CAUCGGCCGC CGAUAUUGGC

In other embodiments, the present invention also contemplates methodsfor detecting the presence of Neisseria gonorrhoeae microorganisms bycontacting a test sample under stringent hybridization conditions with anucleic acid hybridization assay probe capable of preferentiallyhybridizing under stringent hybridization assay conditions to aNeisseria gonorrhoeae nucleic acid sequence over nucleic acid sequencesfrom Neisseria meningitidis, said target nucleic acid sequencessubstantially corresponding to a sequence selected form the groupconsisting of:

SEQ ID NO 1: GAACGTACCG GGTAGCGG

SEQ ID NO 3: GCCAATATCG GCGGCCGATG

SEQ ID NO 31: GAACGUACCG GGUAGCGG

SEQ ID NO 32: GCCAAUAUCG GCGGCCGAUG

The present invention also contemplates methods of detecting Neisseriaby first amplifying a portion of the Neisseria nucleic acid and thenoptionally using a hybridization assay probe of the present invention toassay for a specific Neisseria-derived nucleic acid amplified by theprimers of the present invention. The amplified nucleic acid can bedetected by a number of methods including gel electrophoresis.

In preferred embodiments, the present invention contemplates methods ofdetecting Neisseria-derived nucleic acid by first amplifying saidnucleic acid with at least one amplification oligonucleotide that willbind to or cause elongation through one or more of the followingsequences:

SEQ ID NO: 47 GUCUUGAGAG GGAAAGCAGG GGAC

SEQ ID NO: 48 UAUGUUACUC ACCCGUUCGC CACUCGCC

SEQ ID NO: 49 CUGAAGAAUA AGCACCGGCU AACUACGUGC AGCAGC

SEQ ID NO: 50 CGAUGACGGU ACCUGAAGAA UAAGCACCGG CUAAC

SEQ ID NO: 51 AACGGCCUUU UCUUCCCUGA CAAAAGUCCU UUACAACCCG

SEQ ID NO: 52 GUCCUUUACA ACCCGAAGGC CUUC

wherein said amplification oligonucleotide optionally has a nucleic acidsequence recognized by an RNA polymerase or which enhances initiation ofelongation by an RNA polymerase.

This first method step is then optionally followed by detecting theamplified nucleic acid produced in the amplification step with anoligonucleotide hybridization assay probe able to specifically hybridizeto nucleic acids derived from Neisseria species, Neisseria cinerea,Neisseria meningitidis or Neisseria gonorrhoeae under stringenthybridization conditions.

The amplification oligonucleotide used in the methods of the presentinvention may optionally have a nucleic acid sequence for example, apromoter sequence, recognized by an RNA polymerase or which enhancesinitiation by an RNA polymerase.

In other preferred embodiments, the present invention contemplates amethod for amplifying Neisseria nucleic acids in a test sample byamplifying the nucleic acid with one or more amplificationoligonucleotide that will bind to or cause elongation through a nucleicacid sequence substantially corresponding to the following nucleotidesequences:

SEQ ID NOs: 19 or 49,

SEQ ID NOs: 21 or 50, or

with a second amplification oligonucleotide that will bind to or causeelongation through a nucleic acid sequence substantially correspondingto one of the following sequences:

SEQ ID NOs: 20 or 51,

SEQ ID NOs: 22 or 52 or both, said amplification oligonucleotides,wherein at least one of said amplification oligonucleotides optionallyhas a nucleic acid sequence recognized by an RNA polymerase or whichenhances initiation or elongation by an RNA polymerase.

In other more preferred embodiments, the present invention contemplatesa method for amplifying Neisseria-derived nucleic acids in a test samplecomprising amplifying said nucleic acid with one or more amplificationoligonucleotide that will bind to or cause elongation through a nucleicacid sequence substantially corresponding to one of the followingnucleotide sequences:

SEQ ID NOS: 19 or 49, or

with a second amplification oligonucleotide that will bind to or causeelongation through a nucleic acid sequence substantially correspondingto the following sequences: SEQ ID NOS: 20 or 51, or both saidamplification oligonucleotides, wherein at least one of saidamplification oligonucleotides optionally has a nucleic acid sequencerecognized by an RNA polymerase or which enhances initiation orelongation by an RNA polymerase.

In other preferred embodiments, the present invention contemplates amethod for increasing the number of Neisseria-derived nucleic acidsequences in a test sample comprising amplifying said nucleic acid withone or more amplification oligonucleotides that will bind to or causeelongation through a nucleic acid sequence substantially correspondingto the following nucleotide sequences:

SEQ ID NOS: 21 or 50,

or with a second amplification oligonucleotide that will bind to orcause elongation through a nucleic acid sequence substantiallycorresponding to the following sequences:

SEQ ID NOS: 22 or 52, or both said amplification oligonucleotides,wherein at least one of said amplification oligonucleotides optionallyhas a nucleic acid sequence recognized by an RNA polymerase or whichenhances initiation or elongation by an RNA polymerase.

The above methods may also include the further step of detecting theamplified nucleic acid with an oligonucleotide hybridization assay probeable to specifically hybridize to Neisseria meningitidis nucleic acidsunder stringent hybridization conditions.

Specifically, the methods may detect Neisseria meningitidis usingoligonucleotide hybridization assay probes which will hybridize understringent hybridization conditions to a nucleic acid sequencesubstantially corresponding to a sequence selected from the groupconsisting of:

SEQ ID NO: 11 GGCTGTTGCT AATATCAGCG

SEQ ID NO: 27 CGCUGAUAUU AGCAACAGCC

SEQ ID NO: 12 GGCTGTTGCT AATACCAGCG

SEQ ID NO: 28 CGCUGGUAUU AGCAACAGCC

SEQ ID NO: 15 CGCTGATATT AGCAACAGCC

SEQ ID NO: 25 GGCUGUUGCU AAUAUCAGCG

SEQ ID NO: 16 CGCTGGTATT AGCAACAGCC

SEQ ID NO: 26 GGCUGUUGCU AAUACCAGCG

The present invention also contemplates methods for increasing thenumber of Neisseria gonorrhoeae-derived nucleic acids in a test sampleby amplifying said nucleic acid with one or more amplificationoligonucleotides that will bind to or cause elongation through a nucleicacid sequence substantially corresponding to one or more of thefollowing nucleotide sequences:

SEQ ID NOs 23 or 47,

SEQ ID NOs 24 or 48,

and where the amplification oligonucleotide optionally has a nucleicacid sequence recognized by an RNA polymerase or which enhancesinitiation or elongation by an RNA polymerase.

Additional methods are contemplated for amplifying Neisseriagonorrhoeae-derived nucleic acids in a test sample with a firstamplification oligonucleotide that will bind to or cause elongationthrough a nucleic acid sequence substantially corresponding to one ofthe following nucleotide sequences:

SEQ ID NOs: 23 or 47, or

with a second amplification oligonucleotide that will bind to or causeelongation through a nucleic acid sequence substantially correspondingto one of the following sequences:

SEQ ID NOs: 24 or 48, or

with both said first and second amplification oligonucleotides whereinone of the amplification oligonucleotides optionally has a nucleic acidsequence recognized by an RNA polymerase or which enhances initiation orelongation by an RNA polymerase.

These methods of amplifying a Neisseria gonorrhoeae-derived nucleic acidmay be followed by the step of detecting the amplified nucleic acid withan oligonucleotide hybridization assay probe able to specificallyhybridize to Neisseria gonorrhoeae nucleic acids under stringenthybridization conditions.

Preferably the oligonucleotide hybridization assay probe has a nucleicacid sequence substantially corresponding to a sequence selected fromthe group consisting of:

SEQ ID NOS: 1, 29, 31, and 33.

The detecting of Neisseria gonorrhoeae nucleic acid may include the useof a helper oligonucleotide having a nucleic acid sequence substantiallycorresponding to a nucleic acid sequence selected from the groupconsisting of:

SEQ ID NO: 2, and

SEQ ID NO: 39.

Other methods of detecting Neisseria gonorrhoeae nucleic acid arecontemplated by increasing the number of Neisseria gonorrhoeae-derivednucleic acids in a test sample by amplifying said nucleic acid with oneor more amplification oligonucleotide that will bind to or causeelongation through a nucleic acid sequence substantially correspondingto one or more of the following nucleotide sequences: SEQ ID NOS: 19 or49, SEQ ID NOS: 20 or 51, SEQ ID NOS: 21 or 50, SEQ ID NOS: 22 or 52,and where the amplification oligonucleotide optionally has a nucleicacid sequence recognized by an RNA polymerase or which enhancesinitiation or elongation by an RNA polymerase.

Preferred methods for amplifying Neisseria nucleic acids in a testsample include amplifying the nucleic acid with one or moreamplification oligonucleotide that will bind to or cause elongationthrough a nucleic acid sequence substantially corresponding to one ofthe following nucleotide sequences:

SEQ ID NOS: 19 or 49, or

with a second amplification oligonucleotide that will bind to or causeelongation through a nucleic acid sequence substantially correspondingto one of the following sequences:

SEQ ID NOS: 20 or 51, or

with both said first and second amplification oligonucleotides.

Alternatively the present invention contemplates amplifying Neisserianucleic acids in a test sample comprising amplifying the nucleic acidwith one or more amplification oligonucleotide that will bind to orcause elongation through a nucleic acid sequence substantiallycorresponding to one of the following nucleotide sequences:

SEQ ID NOS: 21 or 50, or

with a second amplification oligonucleotides that will bind to or causeelongation through a nucleic acid sequence substantially correspondingto one of the following sequences:

SEQ ID NOS: 22 or 50, or

with both said first and second amplification oligonucleotides.

The amplification of the Neisseria nucleic acid is preferably followedby detecting the amplified nucleic acid with an oligonucleotidehybridization assay probe able to specifically hybridize to Neisseriagonorrhoeae nucleic acids under stringent hybridization conditions. Theoligonucleotide hybridization assay probe used preferably has a nucleicacid sequence substantially corresponding to a sequence selected fromthe group consisting of:

SEQ ID NOS: 3, 32, 36, 34

H. Diagnostic Systems

The present invention also contemplates diagnostic systems in kit form.A diagnostic system of the present invention may include a kit whichcontains, in an amount sufficient for at least one assay, amplificationprimers and/or hybridization assay probes of the present invention in apackaging material. Typically, the kits would also include instructionsfor use of the packaged primers and/or probes.

The various components of the diagnostic system may be provided invarious forms. For example, the required enzymes, the nucleotidetriphosphates, the primers and probes may be provided as a lyophilizedreagent. These lyophilized reagents may be premixed beforelyophilization so that when reconstituted form a complete mixture withthe proper ratio of each of the components ready for use in the assay.In addition, the diagnostic systems of the present invention may containa reconstitution reagent for reconstituting the lyophilized reagents ofthe kit. In preferred kits, the enzymes, nucleotides, triphosphates andrequired cofactors for the enzymes are provided as a single lyophilizedreagent that when reconstituted forms a proper reagent for use in thepresent methods. In these preferred kits, a lyophilized primer agent mayalso be provided. In other preferred kits, lyophilized probe reagentsare provided.

Typical packaging materials would include solid matrices such as glass,plastic, paper, foil, micro particles and the like, capable of holdingwithin fixed limits hybridization assay probe or amplification primer ofthe present invention. Thus, for example, a package made from packagingmaterials can be a glass vial used to contain sub-milligram (i.e.picogram, nanogram etc.) quantities of a contemplated primer orhybridization assay probe or it could be a microtiter plate well towhich the probes and/or primers of the present invention have beenoperatively affixed, i.e., linked so. as to be capable of participatingin a detection method of the present invention.

Instructions for use typically include a tangible expression describingthe various reagents and/or concentrations of reagents and at least oneassay method parameter which, for example, would be the relative amountsof reagents to use per amount of sample. In addition, such specifics asmaintenance, time periods, temperature and buffer conditions may also beincluded.

The present invention contemplates diagnostic systems or kits containingthe oligonucleotides of a composition of the present invention. Thepresent invention also contemplates diagnostic systems or kitscontaining the oligonucleotides required to perform a method of thepresent invention.

This method preferably also uses a helper oligonucleotide having anucleic acid sequence substantially corresponding to a nucleic acidsequence selected from the group consisting of:

SEQ ID NO. 4: and

SEQ ID NO. 40:

The present invention contemplates diagnostic systems or a kitcontaining at least one oligonucleotide having a nucleic acid sequencesubstantially corresponding to a nucleic acid sequence selected from thegroup consisting of:

SEQ ID NOS: 1, 3, 11, 12, 15, 16, 29, 30, 33, 34, 27, 28, 25, 26.

The present invention contemplates diagnostic systems or a kit having anoligonucleotide hybridization assay probe having at least one helperprobe having a nucleic acid sequence substantially corresponding to thesequence selected from the group consisting of:

SEQ ID NOS: 2 or 39,

when said oligonucleotide substantially corresponds to

SEQ ID NOS: 1 or 31; or

SEQ ID NOS: 4 or 40,

when said oligonucleotide substantially corresponds to

SEQ ID NOS: 3 or 32; or

SEQ ID NOS: 13 or 35, or

SEQ ID NOS: 14 or 36,

when said oligonucleotide substantially corresponds to

SEQ ID NOS: 11 or 25, or

SEQ ID NOS: 12 or 26; or

SEQ ID NOS: 17 or 37,

SEQ ID NOS: 18 or 38,

when said oligonucleotide substantially corresponds to

SEQ ID NOS: 15 or 27, or

SEQ ID NOS: 16 or 28.

The present invention contemplates diagnostic systems or a kitcontaining two oligonucleotides having a nucleic acid sequencesubstantially corresponding to a nucleic acid sequence selected from thegroup consisting of:

SEQ ID NO: 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG

SEQ ID NO: 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG

SEQ ID NO: 9 CGGGTTGTAA AGGACTTTTG TCAGGGAAGA AAAGGCCGTT

SEQ ID NO: 10 GAAGGCCTTC GGGTTGTAAA GGAC

SEQ ID NO: 43 GCUGCUGCAC GUAGUUAGCC GGUGCUUAUU CUUCAG

SEQ ID NO: 44 GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG

SEQ ID NO: 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU

SEQ ID NO: 46 GAAGGCCUUC GGGUUGUAAA GGAC

optionally having a 5′ sequence recognized by an RNA polymerase or whichenhances initiation or elongation by an RNA polymerase.

The present invention contemplates diagnostic systems or a kitcontaining oligonucleotides having a nucleic acid sequence substantiallycorresponding to the following sequences:

SEQ ID NOS: 7 or 43,

SEQ ID NOS: 9 or 45,

SEQ ID NOS: 11 or 25,

SEQ ID NOS: 13 or 35,

SEQ ID NOS: 14 or 36.

The present invention contemplates diagnostic systems or a kitcontaining oligonucleotides having a nucleic acid sequence substantiallycorresponding to the following sequences:

SEQ ID NOS: 15 or 27,

SEQ ID NOS: 16 or 26,

SEQ ID NOS: 17 or 37,

SEQ ID NOS: 18 or 38.

The present invention contemplates diagnostic systems or a kitcontaining oligonucleotides having a nucleic acid sequence substantiallycorresponding to the following sequences:

SEQ ID NOS: 7 or 43,

SEQ ID NOS: 9 or 45,

SEQ ID NOS: 15 or 27,

SEQ ID NOS: 16 or 28,

SEQ ID NOS: 17 or 37,

SEQ ID NOS: 18 or 38.

The present invention contemplates diagnostic systems or a kitcontaining oligonucleotides having a nucleic acid sequence substantiallycorresponding to the following sequences:

SEQ ID NOS: 5 or 41,

SEQ ID NOS: 6 or 42,

SEQ ID NOS: 2 or 39,

SEQ ID NOS: 1 or 31.

The present invention contemplates diagnostic systems or a kitcontaining oligonucleotides having a nucleic acid sequence substantiallycorresponding to the following sequences:

SEQ ID NOS: 5 or 41,

SEQ ID NOS: 2 or 39,

SEQ ID NOS: 1 or 31.

EXAMPLES

Examples are provided below to illustrate different aspects andembodiments of the present invention. These examples are not intended inany way to limit the disclosed invention, which is limited only by theclaims.

Probes specific for Neisseria meningitidis were designed using sequencesdetermined in prospective target areas using primers complementary tothe 16S rRNAs of Neisseria gonorrhoeae (ATCC NO. 19424), Neisseriameningitidis sero group A (ATCC NOs 13077), serogroup C (ATCC No. 23248)and serogroup L (ATCC No. 43828), and clinical isolates, Neisserialactamica (ATCC NO. 23970), Neisseria cinerea (ATCC NO. 14685),Neisseria mucosa (ATCC NO. 19696), Neisseria sicca (ATCC NO. 29193) andKingella kingae (ATCC NO. 23330). The nucleic acid sequence fromphylogenetically near neighbors, including the published sequence ofNeisseria gonorrhoeae NCTC 8375 Rossau et al., Nuc. Acids Res. 16:6227were also used as comparisons with the nucleic sequence from Neisseriameningitidis to determine variable regions.

An example of such an alignment follows: A specific sequence in whichNeisseria meningitidis varied from E. coli and Neisseria gonorrhoeae waschosen for probe design. Two different probes were designed to Neisseriameningitidis (SEQ ID NO: 11) and (SEQ ID NO: 12). The rRNA sequences areshown below:

E. coli GAGUAAAG(UUAAUAC)CUUUG SEQ ID NO: 54 .      .   .. .. .   .GGCTGTTG(CTAATAC)CAGCG SEQ ID NO: 12 ........ ......  .....GGCTGTTG(CTAATAT)CAGCG SEQ ID NO: 11 ... .  . . .. .  ..... N.meningitidis.c GGCUGUUG(CUAAUAU)CAGCG SEQ ID NO: 55........ . ..... . . . N. gonorrhoeae.P GGCUGUUG(CCAAUAU)CGGGG SEQ IDNO: 56

The following hybridization assay probe sequences are featured in theexamples described below:

SEQ ID NO 1: GAACGTACCG GGTAGCGG

SEQ ID NO 3: GCCAATATCG GCGGCCGATG

SEQ ID NO: 11 GGCTGTTGCTAATATCAGCG,

SEQ ID NO: 12 GGCTGTTGCTAATACCAGCG,

SEQ ID NO: 15 CGCTGATATTAGCAACAGCC, and

SEQ ID NO: 16 CGCTGGTATTAGCAACAGCC

Example 1

In this experiment, purified N. gonorrhoeae rRNA (ATCC NO. 19424) wasamplified with oligonucleotides containing sequences complementary to N.gonorrhoeae rRNA using the techniques described in Kacian et al. U.S.Pat. No. 5,399,491. Two promoter primers were synthesized, eachcontaining a T7 RNA polymerase promoter sequence5′-AATTTAATACGACTCACTATAGGGAGA-3′ SEQ ID NO. 53 at the 5′ end,covalently attached to a target complementary sequence5′-GTCCCCTGCTTTCCCTCTCAAGAC-3′ (SEQ ID NO. 5) at the 3′ end. Onepromoter primer was synthesized with a free 3′ OH group, and was used attwo pmol per reaction. The second promoter primer was synthesized withan alkane diol group at the 3′ end and was used at 13 pmol per reaction.The target nucleic acid and primers were heated to 95° C. for 15 minutesand cooled to 42° C. Moloney Murine Leukemia Virus reverse transcriptase(MMLV RT), 900 units, and 400 units of T7 RNA polymerase were added. Thefinal amplification mixture contained 50 mM Tris HCl (pH 8.5), 35 mMpotassium chloride, 4 mM GTP, 4 mM ATP, 4 mM UTP, 4 mM CTP, 1 mM dATP, 1mM dTTP, 1 mM dCTP, 1 mM dGTP, 20 mM MgCl₂, 20 mM N-Acetyl-L-Cysteine,and 5% (w/v) glycerol. After a two hour incubation at 42° C., the entireone hundred μl amplification reaction was assayed by hybridization withan acridinium ester labeled probe of sequence 5′-GAACGTACCGGGTAGCGG-3′(SEQ. ID. NO. 1) and an unlabeled helper probe of sequence5′-GGGATAACTGATCGAAAGA-TCAGCTAATACCGCATACG-3′ (SEQ. ID. NO. 2)Hybridization was performed in 200 μl of a solution containing 0.05 Mlithium succinate (pH 5), 0.6 M LiCl, 1% (w/v) lithium lauryl sulfate,10 mM EDTA, 10 mM EGTA, at 60° C. for 10 minutes, followed by additionof 300 μl of 0.15 M sodium tetraborate pH 8.5, 1% TRITON® X-100. Thismixture was incubated at 60° C. for 10 minutes, and cooled to roomtemperature. The remaining chemiluminescence in each tube was assayed ina Gen-Probe LEADER® I luminometer equipped with automatic injection of 1mM nitric acid and 0.1% (v/v) hydrogen peroxide followed by injection ofa solution containing 1 N sodium hydroxide. Results were given inRelative Light Units (RLU), a measure of the photons detected by theluminometer.

TABLE 1 Amplification of Neisseria gonorrhoeae nucleic acid withamplification oligonucleotides comprising SEQ ID NO. 5 followed bydetection with a probe comprising SEQ ID NO. 1. Amount of target RLU 0.1pg* 103,596 99,931 123,512 0.025 pg 25,636 39,454 29,594 0 pg 1,0841,012 640 *pg = picogram

Example 2

This experiment demonstrates amplification of N. gonorrhoeae rRNA withtwo primers of opposite sense. The promoter-primer described in Example1 containing a T7 RNA polymerase promoter sequence and a 3′ targethybridizing region of sequence 5′-GTCCCCTGCTTTCCCTCTCAAGAC-3′ (SEQ. ID.NO. 5) was used at 15 pmol per reaction and a primer containing asequence of the same sense as N. gonorrhoeae rRNA,5′-GGCGAGTGGCGAACGGGTGAGTAACATA-3′ (SEQ. ID. NO. 6) was used at 15 pmolper reaction. Reactions were performed in triplicate. The amplificationconditions were as described in Example 1, and samples were heated to95° C. for 5 minutes, then cooled to 42° C. Enzymes were added, andafter a two hour incubation at 42° C., 20 μl of the amplificationreaction was assayed by hybridization with an acridinium ester labeledprobe synthesized with sequence SEQ. ID. NO. 1 and an unlabeled helperprobe synthesized with sequence SEQ. ID. NO. 2. The primers amplified N.gonorrhoeae RNA and allowed detection of less than 100 copies of target.

TABLE 2 Amplification of N. gonorrhoeae rRNA with primers comprisingSEQ. ID. NOs. 5 and 6 and detection with a probe comprising SEQ IDNO. 1. Amount of rRNA target added RLU 500 copies (0.0012 pg) 334,838343,107 1,320,194 80 copies (0.0002 pg) 255,898 1,512,486 377,938 0 pg2,354 2,454 2,440

Example 3

In this experiment, two promoter primers of identical sequence wereagain used. Each promoter primer was synthesized with a 5′ T7 RNApolymerase promoter sequence 5′-AATTTAATACGACTCACTATAGGGAGA-3′ SEQ IDNO: 53 at the 5′ end and a target hybridizing region5′-GCTGCTGCACGTAGTTAG-CCGGTGCTTATTCTTCAG-3′ (SEQ ID NO. 7) at the 3′end. The promoter primers were synthesized either with a 3′-hydroxylgroup and used at 2 pmol per reaction, or with a 3′-alkane diol and usedat 13 pmol per reaction. Samples were heated to 95° C. for 5 minutes andcooled to 42° C. prior to enzyme addition. The amplification conditionswere as described in Example 1. After a two hour incubation at 42° C.,100 μl of the amplification reaction was assayed by hybridization withan acridinium ester labeled probe synthesized with sequence5′GCCAATATCGGCGGCCGATG-3′ (SEQ. ID. NO. 3) and an unlabeled helper probewith the sequence 5′-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3′ (SEQ. ID.NO. 4) using the conditions described in Example 1.

TABLE 3 Amplification of N. qonorrhoeae rRNA using primers comprisingSEQ ID NO. 7. Amount of rRNA target added RLU with probe SEQ ID NO. 30.025 pg 95,905 49,717 59,774 0.0125 pg 10,520 12,576 12,322 0.005 pg19,498 40,962 21,722 0 pg 2,888 2,792 2,777

Example 4

In this experiment, N. gonorrhoeae rRNA was amplified with a mixture oftwo oligonucleotides, one a promoter primer complementary to N.gonorrhoeae rRNA and one primer of the same sense as N. gonorrhoeaerRNA. The promoter primer contained a T7 RNA polymerase promotersequence at the 5′ end and a target hybridizing region5′-GCTGCTGCACG-TAGTTAGCCGGTGCTTATTCTTCAG-3′ (SEQ ID NO. 7) at the 3′ endand was used with a primer of sequence5′-CGGGTTGTAAAGGAC-TTTTGTCAGGGAAGAAAAGGCCGTT-3′ (SEQ. ID. NO. 9) at 30pmol per reaction. Alternatively, a promoter primer containing a targethybridizing region of sequence 5′-GTTAGCCGGTGCTTATTCTTCAGGTACCGTCATCG-3′(SEQ. ID. NO. 8) was used at 15 pmol per reaction with the promoterprimer with a sequence 5′-GAAGGCCTTCGGGTTGTAAAGGAC-3′ (SEQ. ID. NO. 10),at 15 pmol per reaction. Amplification conditions were as described forExample 1. Twenty μl of the product was assayed by hybridization with anacridinium ester labeled probe synthesized with sequence5′-GCCAATATCGGCGGCCGATG-3′ (SEQ. ID. NO. 3) and an unlabeled helperprobe synthesized with the sequence5′-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3′ (SEQ. ID. NO. 4) as describedin Example 1.

TABLE 4 Amplification of N. gonorrhoeae rRNA using primers comprisingSEQ ID NOs. 7 and 9 or 8 and 10. RLU with probe SEQ ID NO. 3 Amount ofrRNA target SEQ ID SEQ ID added NOS. NOS. Primer sequences: 7 and 9 8and 10 0.5 pg 817,344 680,836 802,901 603,811 785,206 619,969 0.05 pg188,661 132,359 192,656 157,509 204,878 87,161 0.005 pg 25,276 51,39826,451 40,032 24,915 55,968 0 pg 3,600 2,189 3,366 2,205 888

Example 5

This example demonstrates the reactivity of the amplification anddetection assay. Fresh cultures of thirteen strains of N. gonorrhoeaewere suspended in 0.9% sodium chloride at a density of approximately10¹⁰ cells/ml and lysed in a solution containing 3% (w/v) lithium laurylsulfate, 30 mM sodium phosphate pH 6.8, 1 mM EDTA and 1 mM EGTA. Releaseof nucleic acid was confirmed by hybridization with a probe directed toa conserved region of ribosomal RNA of all bacteria. The cell lysateswere further diluted in water and added to amplification reactionscontaining 30 pmol of a promoter-primer containing a 5′ T7 RNA promotersequence SEQ ID NO: 53 5′-AATTTA-ATACGACTCACTATAGGGAGA-3′ and a 3′target binding sequence comprising SEQ. ID. NO. 7, and 30 pmol of primercomprising sequence SEQ. ID. NO. 9. Duplicate reactions containinglysate from at least 10⁵ cells were performed using an amplificationmixture containing 50 mM Tris HCl (pH 8.5), 35 mM potassium chloride, 4mM GTP, 4 mM ATP, 4 mM UTP, 4 mM CTP, 1 mM DATP, 1 mM dTTP, 1 mM dCTP, 1mM dGTP, 20 mM MgCl₂, 20 mM N-acetyl-L-cysteine, 5% (v/v) glycerol andthe oligonucleotide primers described above. The mixture was heated to95° C. for 5 minutes, cooled to 42° C. and 900 units of MMLV reversetranscriptase and 400 units of T7 RNA polymerase were added. After a onehour incubation at 42° C., 20 μl of amplification reaction was assayedby hybridization with an acridinium ester labeled probe synthesized withsequence 5′GCCAATATCGGCGGCCGATG-3′ (SEQ. ID. No. 3) and an unlabeledhelper probe containing sequence5′-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3′ (SEQ. ID. NO. 4).

TABLE 5 Amplification of different strains of N. gonorrhoeae usingprimers comprising SEQ ID NOs. 7 and 9. N. gonorrhoeae ATCC No. RLU withprobe SEQ ID NO. 3 9793 1,150,477 1,162,284 9826 1,173,586 1,149,2519827 1,093,440 1,080,405 9828 1,143,960 1,149,465 9830 1,165,1081,143,063 10150 1,105,754 1,131,598 10874 1,139,487 1,103,912 116881,024,195 1,112,160 11689 1,141,404 1,116,069 19424 1,104,256 1,116,83227628 1,133,696 1,117,624 27630 1,132,496 1,146,161 27631 1,089,1051,070,058

Example 6

Sequence analysis of other Neisseria species indicated that theamplification oligonucleotides of this invention could amplify nucleicacids of other species. This example demonstrates the utility of theamplification oligonucleotides of this invention to amplify nucleic acidfrom another Neisseria species, N. meningitidis. In the course ofdevelopment of a specific probe for N. meningitidis, it became clearthat the members of the species N. meningitidis were not homogeneous inthe probe region of choice. The sequences of 16S rRNAs of representativeN. meningitidis species which showed low reactivity to the initial probewere determined and a second probe was designed. These data demonstratethe differential reactivity of three N. meningitidis species to the twoprobes. In this example, purified RNA from Neisseria gonorrhoeae (ATCCNo. 19424), or lysates from Neisseria meningitidis serogroup A (ATCC No.13077), serogroup C (ATCC No. 13102) and serogroup L, (ATCC No. 43828)representing approximately 1,000 cells were amplified with apromoter-primer and primer described in Example 5 under the conditionsdescribed in Example 5. Ten μl samples of the 100 μl amplificationreactions were assayed by hybridization with an acridinium ester labeledprobe synthesized with sequence 5′-GCCAATATCGGCGGCCGATG-3′ (SEQ ID NO. 3and an unlabeled helper probe synthesized with the sequence5′-ACGGTACCTGAAGAATAAGCACCGGCTAACTACGTG-3, (SEQ ID NO. 4), or anacridinium ester labeled probe synthesized with the sequence5′-GGCTGTTGCTAATATCAGCG-3′ (SEQ ID NO. 11) and two unlabeled helperprobes, one synthesized with sequence5′-GCCTTCGGGTTGTAAAGGACTTTTGTCAGGGAAGAAAA-3′ (SEQ ID NO. 13) and onesynthesized with the sequence 5′-GCTGATGACGGTACCTGAAGAATAAGCACCGGC-3′(SEQ ID NO. 14), or an acridinium ester labeled probe synthesized withsequence 5′-GGCTGTTGCTAATACCAGCG-3′ (SEQ ID NO. 12) with unlabeledhelper probes SEQ ID NO: 13 and 14 or with a combination of labeledprobes SEQ ID NO: 11 and 12 used with unlabeled helper probes SEQ ID NO:13 and 14. Sequence analysis indicated that other strains of Neisseriawill also amplify with these primers.

TABLE 6 Amplification of Neisseria gonorrhoeae and Neisseriameningitidis strains using primers comprising SEQ ID NOs. 7 and 9. RLUProbe SEQ ID NOs: 3 11 12 11 + 12 Helper probe 4 13 + 14 13 + 14 13 + 14SEQ ID NOs: Organism N. gonorrhoeae 1,017,626 1,660 820 1,603 994,7881,448 809 1,559 1,030,242 1,743 805 1,792 N. meningitidis 2,0591,208,967 3,534 829,251 Serogroup A 1,861 1,115,956 3,700 760,360 2,1831,138,675 3,546 775,675 N. meningitidis 1,931 1,164,254 2,819 749,502Serogroup C 2,130 1,068,489 2,477 687,517 1,963 1,110,933 3,103 803,732N. meningitidis 1,833 85,321 1,206,045 1,537,314 Serogroup L 1,97279,555 1,199,815 1,474,016 1,814 77,797 1,211,022 1,645,742

The data show that strains of N. meningitidis and N. gonorrhoeae can beamplified using primers comprising SEQ ID NOs. 7 and 9 and detected withprobes of SEQ ID NOs. 3, 11, and 12.

Example 7

The sensitivity of the amplification and detection assay for N.meningitidis were demonstrated in this experiment. In this example,Neisseria meningitidis serogroup C cells were cultured and suspended in0.9% sodium chloride to a density of approximately 10⁹ cells per ml.Cells were lysed following addition of an equal volume of a solutioncontaining 3% (w/v) lithium lauryl sulfate, 30 mM sodium phosphate (pH6.8), 1 mM EDTA, 1 mM EGTA and diluted with water prior to addition tothe amplification reactions. Amplifications were performed as describedfor Example 5 using the promoter primer and primer described in Example5 (SEQ ID NOs. 7 and 9, respectively). Twenty μl of the reaction wasanalyzed by hybridization in the HPA format using an acridinium esterlabeled probe synthesized with the sequence 5′-GGCTGTTGCTAATATCAGCG-3′(SEQ ID NO. 11) and two unlabeled helper probes, one synthesized withthe sequence 5′-GCCTTCGGGTTG-TAAAGGACTTTTGTCAGGGAAGAAAA-3′ (SEQ ID NO.13) and one synthesized with the sequence5′-GCTGATGACGGTACCTGAAGAATAAGCACCGGC-3′ (SEQ ID NO. 14).

TABLE 7 Amplification of N. meningitidis serogroup A with amplificationoligomers comprising SEQ ID NOs. 7 and 9, followed by detection withprobe SEQ ID NO. 11. RLU with Amount of probe SEQ ID target added NO. 1140 cells 723,645 648,069 686,492 4 cells 195,370 189,451 162,128 0.4cells 28,585 23,253 824,742 64,945 0 cells 1,432 1,202 1,258

Example 8

To demonstrate the reactivity and specificity of the probes directed toN. meningitidis 16S rRNA, a mixture of probes containing acridiniumester labeled oligonucleotides synthesized with the sequence5′-CGCTGATATTAGCAACAGCC-3′, (SEQ ID NO. 15) or sequence5′-CGCTGGTATTAGCAACAGCC-3′, (SEQ ID NO. 16), and unlabeled helper probessynthesized with the sequence5′-TTTTCTTCCCTGACAAAAGTCCTTTACAACCCGAAGGC-3′ (SEQ ID NO. 17 and5′-GCCGGTGCTTATTCTTCAGGTACCGTCATCAG-3′ (SEQ ID NO. 18), were hybridizedto nucleic acid in lysates prepared from fresh cultures of the Neisseriaspecies listed below. Each lysate was tested with a probe directed to aconserved region of 23S rRNA to confirm the lysis of the organism andintegrity of the rRNA.

TABLE 8 Reactivity and specificity of probes directed to N. meningitidis16S rRNA. RLU with ATCC RLU with conserved Organism No. probe mix* probeN. cinerea 14685 736,927 59,831 N. denitrificans 14686 581 50,391 N.elongata 25295 1,511 52,017 N. elongata subspe- 29315 618 53,312 ciesglycolytica N. flavescens 13120 1,316 53,397 N. gonorrhoeae 9793 1,82662,658 N. gonorrhoeae 9827 753 60,252 N. gonorrhoeae 9830 4,832 58,346N. gonorrhoeae 10150 1,139 61,573 N. gonorrhoeae 10874 759 58,291 N.gonorrhoeae 11689 4,824 60,039 N. gonorrhoeae 19088 910 53,594 N.gonorrhoeae 19424 851 60,372 N. gonorrhoeae 21824 746 62,153 N.gonorrhoeae 27630 1,829 53,241 N. gonorrhoeae 33084 784 62,696 N.gonorrhoeae 35541 431 59,229 N. lactamica 23970 3,497 54,255 N.meningitidis ser- 13077 844,739 54,292 ogroup A N. meningitidis ser-23255 722,108 61,439 ogroup B N. meningitidis ser- 13090 704,890 57,321ogroup B N. meningitidis ser- 23251 761,475 58,545 ogroup B N.meningitidis ser- 13103 770,221 63,704 ogroup C N. meningitidis ser-13106 761,099 60,928 ogroup C N. meningitidis ser- 13102 752,743 62,351ogroup C N. meningitidis ser- 13111 711,196 59,635 ogroup C N.meningitidis ser- 13109 768,874 63,295 ogroup C N. meningitidis ser-13110 676,060 58,150 ogroup C N. meningitidis ser- 13112 543,492 54,921ogroup C N. meningitidis ser- 23248 321,600 59,308 ogroup C N.meningitidis ser- 13113 770,893 56,429 ogroup D N. meningitidis 35558797,072 58,882 group E N. meningitidis ser- 43828 559,406 61,534 ogroupL N. meningitidis ser- 43744 705,798 62,152 ogroup W-135 N. meningitidisser- 35561 778,600 54,938 ogroup Y N. meningitidis ser- 35562 749,75661,793 ogroup Z N. meningitidis 13095 726,612 52,614 N. meningitidis13101 775,912 59,839 N. meningitidis 13804 785,737 61,790 N.meningitidis 43743 734,400 61,357 N. mucosa 19696 1,560 53,427 N. mucosasubspecies 25999 1,761 59,306 heidelbergensis N. sicca 29193 1,20558,260 N. sicca 9913 2,203 57,764 N. subflava 14799 2,046 50,832Negative sample 5,251 124 467 132 1,691 138 *probe mix containedacridinium ester labeled probes synthesized with sequences of SEQ ID NO.15 and SEQ ID NO. 16 and unlabeled helper probes synthesized withsequences of SEQ ID NO. 17 and SEQ.ID NO. 18.

The data show that the mixture of probes allowed detection of all of theN. meningitidis strains tested. The probe mix did show a cross reactionwith N. cinerea, an organism unlikely to be found in the same clinicalsamples as N. meningitidis. Treatment of patients with N. cinereainfections would be the same as for patients infected with N.meningitidis.

Example 9

This example demonstrates the specificity of the amplification anddetection assay. Thirty pmol of the promoter-primer comprising SEQ. ID.NO. 7 and 30 pmol of the primer comprising SEQ. ID. NO. 9 were used inthe assay with eleven different Neisseria species. Cell lysates wereprepared as described in Example 5 and amplified and analyzed byhybridization using the conditions described in Example 1. Twentymicroliters of the amplification reactions were hybridized to anacridinium ester labeled probe synthesized with sequence5′-GCCAA-TATCGGCGGCCGATG -3′ (SEQ ID NO. 3) and an unlabeled helperprobe synthesized with the sequence5′-ACGGTACCTGAAGAATA-AGCACCGGCTAACTACGTG-3′ (SEQ ID NO. 4), or anacridinium ester labeled probe synthesized with the sequence5′-GGCTGTTGCTAATATCAGCG-3′ (SEQ ID NO. 11) in the presence of unlabeledhelper probes synthesized with sequences comprising SEQ ID NOs. 13 and14, or an acridinium ester labeled probe synthesized with the sequence5′-GGCTGTTGC-TAATACCAGCG-3′ (SEQ ID NO. 12) in the presence of unlabeledhelper probes of SEQ ID NOs. 13 and 14.

TABLE 9 Specificity of an assay using amplification witholigonucleotides comprising SEQ ID NOs. 7 and 9 followed by detectionwith probes comprising SEQ ID NOs. 3, 11 or 12. RLU SEQ ID NOs.: ProbeProbe to 11 12 3 conserved regions of bacterial rRNA Helpers 13 + 1413 + 14 4 Organism ATCC No. Neisseria 14685 2,468,721 540,699 1,8041,633 cinerea 609,648 2,484 1,536 575,050 1,943 1,494 Neisseria 146862,339,034 740 644 1,563 denitrificans 659 578 1,539 Neisseria 252952,486,745 772 428 1,521 elongata 738 3,297 1,528 Neisseria 293152,397,697 697 431 1,443 elongata 954 813 1,528 subspecies glycolyticaNeisseria 13120 2,622,452 780 493 1,547 flavescens 874 481 1,610 969 4291,589 Neisseria 23970 2,299,619 736 410 1,621 lactamica 839 425 1,5441,583 428 1,559 Neisseria 19696 2,565,699 1,021 981 1,596 mucosa 1,408559 6,781 851 5,260 1,574 Neisseria 25999 2,927,147 653 367 1,430 mucosa664 390 1,971 heidelber- gensis Neisseria 9913 2,427,561 699 777 1,609sicca 847 477 1,552 834 437 1,642 Neisseria 29193 2,804,642 954 4231,588 sicca 615 388 1,505 Neisseria 19424 N.T. 3,826 419 586,358gonorrhoeae* 1,092 411 564,987 2,390 388 554,134 Neisseria 13077 N.T.557,656 1,287 1,492 meningitidis* 621,180 1,009 1,509 Sero- 539,592 9541,617 group A *purified RNA used at 500 pg per reaction. N.T. = Nottested.

The data shown in the examples described above confirm that the novelamplification oligonucleotides herein described and claimed are capableof amplifying Neisseria nucleic acid and can be used in an assay todistinguish Neisseria meningitidis or Neisseria gonorrhoeae from eachother, the closest known phylogenetic neighbours. None of the examplesdescribed herein are intended to limit the present invention to theembodiments of this disclosure, said invention being limited exclusivelyby the claims which follow.

56 18 base pairs nucleic acid single linear 1 GAACGTACCG GGTAGCGG 18 38base pairs nucleic acid single linear 2 GGGATAACTG ATCGAAAGAT CAGCTAATACCGCATACG 38 20 base pairs nucleic acid single linear 3 GCCAATATCGGCGGCCGATG 20 36 base pairs nucleic acid single linear 4 ACGGTACCTGAAGAATAAGC ACCGGCTAAC TACGTG 36 24 base pairs nucleic acid single linear5 GTCCCCTGCT TTCCCTCTCA AGAC 24 28 base pairs nucleic acid single linear6 GGCGAGTGGC GAACGGGTGA GTAACATA 28 36 base pairs nucleic acid singlelinear 7 GCTGCTGCAC GTAGTTAGCC GGTGCTTATT CTTCAG 36 35 base pairsnucleic acid single linear 8 GTTAGCCGGT GCTTATTCTT CAGGTACCGT CATCG 3540 base pairs nucleic acid single linear 9 CGGGTTGTAA AGGACTTTTGTCAGGGAAGA AAAGGCCGTT 40 24 base pairs nucleic acid single linear 10GAAGGCCTTC GGGTTGTAAA GGAC 24 20 base pairs nucleic acid single linear11 GGCTGTTGCT AATATCAGCG 20 20 base pairs nucleic acid single linear 12GGCTGTTGCT AATACCAGCG 20 38 base pairs nucleic acid single linear 13GCCTTCGGGT TGTAAAGGAC TTTTGTCAGG GAAGAAAA 38 33 base pairs nucleic acidsingle linear 14 GCTGATGACG GTACCTGAAG AATAAGCACC GGC 33 20 base pairsnucleic acid single linear 15 CGCTGATATT AGCAACAGCC 20 20 base pairsnucleic acid single linear 16 CGCTGGTATT AGCAACAGCC 20 38 base pairsnucleic acid single linear 17 TTTTCTTCCC TGACAAAAGT CCTTTACAAC CCGAAGGC38 33 base pairs nucleic acid single linear 18 GCCGGTGCTT ATTCTTCAGGTACCGTCATC AGC 33 36 base pairs nucleic acid single linear 19 CTGAAGAATAAGCACCGGCT AACTACGTGC AGCAGC 36 40 base pairs nucleic acid single linear20 AACGGCCTTT TCTTCCCTGA CAAAAGTCCT TTACAACCCG 40 35 base pairs nucleicacid single linear 21 CGATGACGGT ACCTGAAGAA TAAGCACCGG CTAAC 35 24 basepairs nucleic acid single linear 22 GTCCTTTACA ACCCGAAGGC CTTC 24 24base pairs nucleic acid single linear 23 GTCTTGAGAG GGAAAGCAGG GGAC 2428 base pairs nucleic acid single linear 24 TATGTTACTC ACCCGTTCGCCACTCGCC 28 20 base pairs nucleic acid single linear 25 GGCUGUUGCUAAUAUCAGCG 20 20 base pairs nucleic acid single linear 26 GGCUGUUGCUAAUACCAGCG 20 20 base pairs nucleic acid single linear 27 CGCUGAUAUUAGCAACAGCC 20 20 base pairs nucleic acid single linear 28 CGCUGGUAUUAGCAACAGCC 20 18 base pairs nucleic acid single linear 29 CCGCTACCCGGTACGTTC 18 20 base pairs nucleic acid single linear 30 CATCGGCCGCCGATATTGGC 20 18 base pairs nucleic acid single linear 31 GAACGUACCGGGUAGCGG 18 20 base pairs nucleic acid single linear 32 GCCAAUAUCGGCGGCCGAUG 20 18 base pairs nucleic acid single linear 33 CCGCUACCCGGUACGUUC 18 20 base pairs nucleic acid single linear 34 CAUCGGCCGCCGAUAUUGGC 20 38 base pairs nucleic acid single linear 35 GCCUUCGGGUUGUAAAGGAC UUUUGUCAGG GAAGAAAA 38 33 base pairs nucleic acid singlelinear 36 GCUGAUGACG GUACCUGAAG AAUAAGCACC GGC 33 38 base pairs nucleicacid single linear 37 UUUUCUUCCC UGACAAAAGU CCUUUACAAC CCGAAGGC 38 33base pairs nucleic acid single linear 38 GCCGGUGCUU AUUCUUCAGGUACCGUCAUC AGC 33 38 base pairs nucleic acid single linear 39 GGGAUAACUGAUCGAAAGAU CAGCUAAUAC CGCAUACG 38 36 base pairs nucleic acid singlelinear 40 ACGGUACCUG AAGAAUAAGC ACCGGCUAAC UACGUG 36 24 base pairsnucleic acid single linear 41 GUCCCCUGCU UUCCCUCUCA AGAC 24 28 basepairs nucleic acid single linear 42 GGCGAGUGGC GAACGGGUGA GUAACAUA 28 36base pairs nucleic acid single linear 43 GCUGCUGCAC GUAGUUAGCCGGUGCUUAUU CUUCAG 36 35 base pairs nucleic acid single linear 44GUUAGCCGGU GCUUAUUCUU CAGGUACCGU CAUCG 35 40 base pairs nucleic acidsingle linear 45 CGGGUUGUAA AGGACUUUUG UCAGGGAAGA AAAGGCCGUU 40 24 basepairs nucleic acid single linear 46 GAAGGCCUUC GGGUUGUAAA GGAC 24 24base pairs nucleic acid single linear 47 GUCUUGAGAG GGAAAGCAGG GGAC 2428 base pairs nucleic acid single linear 48 UAUGUUACUC ACCCGUUCGCCACUCGCC 28 36 base pairs nucleic acid single linear 49 CUGAAGAAUAAGCACCGGCU AACUACGUGC AGCAGC 36 35 base pairs nucleic acid single linear50 CGAUGACGGU ACCUGAAGAA UAAGCACCGG CUAAC 35 40 base pairs nucleic acidsingle linear 51 AACGGCCUUU UCUUCCCUGA CAAAAGUCCU UUACAACCCG 40 24 basepairs nucleic acid single linear 52 GUCCUUUACA ACCCGAAGGC CUUC 24 27base pairs nucleic acid single linear 53 AATTTAATAC GACTCACTAT AGGGAGA27 20 base pairs nucleic acid single linear 54 GAGUAAAGUU AAUACCUUUG 2020 base pairs nucleic acid single linear 55 GGCUGUUGCU AAUAUCAGCG 20 20base pairs nucleic acid single linear 56 GGCUGUUGCC AAUAUCGGGG 20

What is claimed is:
 1. A hybridization assay probe for use in detectingthe presence of Neisseria meningitidis in a sample, wherein the basesequence of said probe consists of the base sequence of a first sequenceselected from the group consisting of: SEQ ID NO. 11; SEQ ID NO. 15; SEQID NO. 25; and SEQ ID NO.
 27. 2. The probe of claim 1, wherein saidfirst sequence is SEQ ID NO.
 11. 3. The probe of claim 1, wherein saidfirst sequence is SEQ ID NO.
 15. 4. The probe of claim 1, wherein saidfirst sequence is SEQ ID NO.
 25. 5. The probe of claim 1, wherein saidfirst sequence is SEQ ID NO.
 27. 6. A kit comprising: a hybridizationassay probe for use in detecting the presence of Neisseria meningitidisin a sample, wherein the base sequence of said probe consists of thebase sequence of a first sequence selected from the group consisting of:SEQ ID NO. 11; SEQ ID NO. 15; SEQ ID NO. 25; and SEQ ID NO. 27; and atleast one helper probe, wherein the base sequence of said helper probeconsists of the base sequence of a second sequence selected from thegroup consisting of: SEQ ID NO. 13; SEQ ID NO. 14; SEQ ID NO. 17; SEQ IDNO. 18; SEQ ID NO. 35; SEQ ID NO. 36; SEQ ID NO. 37; and SEQ ID NO. 38.7. The kit of claim 6, wherein: said first sequence is selected from thegroup consisting of: SEQ ID NO. 11; and SEQ ID NO. 25; and said secondsequence is selected from the group consisting of: SEQ ID NO. 13; SEQ IDNO. 14; SEQ ID NO. 35; and SEQ ID NO.
 36. 8. The kit of claim 6,wherein: said first sequence is selected from the group consisting of:SEQ ID NO. 15; and SEQ ID NO. 27; and said second sequence is selectedfrom the group consisting of: SEQ ID NO. 17; SEQ ID NO. 18; SEQ ID NO.37; and SEQ ID NO.
 38. 9. The kit of claim 6, wherein said at least onehelper probe includes first and second helper probes, wherein: saidfirst sequence is SEQ ID NO. 11; said second sequence is SEQ ID NO. 13for said first helper probe; and said second sequence is SEQ ID NO. 14for said second helper probe.
 10. The kit of claim 6, wherein said atleast one helper probe includes first and second helper probes, wherein:said first sequence is SEQ ID NO. 15; said second sequence is SEQ ID NO.17 for said first helper probe; and said second sequence is SEQ ID NO.18 for said second helper probe.
 11. A kit comprising: a hybridizationassay probe for use in detecting the presence of Neisseria meningitidisin a sample, wherein the base sequence of said probe consists of thebase sequence of a first sequence selected from the group consisting of:SEQ ID NO. 11; SEQ ID NO. 15; SEQ ID NO. 25; and SEQ ID NO. 27; and atleast one amplification oligonucleotide, wherein the base sequence ofsaid amplification oligonucleotide consists of the base sequence of asecond sequence selected from the group consisting of: SEQ ID NO. 7; SEQID NO. 9; SEQ ID NO. 43; and SEQ ID NO. 45, or wherein the base sequenceof said amplification oligonucleotide consists of a 3′ base sequencewhich is perfectly homologous to said second sequence and a 5′ basesequence which is recognized by an RNA polymerase or which enhancesinitiation or elongation by an RNA polymerase.
 12. The kit of claim 11,wherein the base sequence of said amplification oligonucleotide consistsof the base sequence of said second sequence.
 13. The kit of claim 11,wherein said second sequence is SEQ ID NO. 7 or SEQ ID NO.
 43. 14. Thekit of claim 11, wherein said second sequence is SEQ ID NO. 9 or SEQ IDNO.
 45. 15. The kit of claim 11, wherein said at least one amplificationoligonucleotide includes first and second amplificationoligonucleotides, wherein: said second sequence is SEQ ID NO. 7 or SEQID NO. 43 for said first amplification oligonucleotide; and said secondsequence is SEQ ID NO. 9 or SEQ ID NO. 45 for said second amplificationoligonucleotide.
 16. The kit of claim 11, wherein said first sequence isSEQ ID NO.
 11. 17. The probe of claim 1 further comprising a detectablelabel.
 18. A composition comprising a nucleic acid hybrid formed betweensaid probe and said first target region of any one of claims 2-5.
 19. Aprobe mix comprising the probe of claim 1 and at least one helper probe,wherein the base sequence of said helper probe consists of the basesequence of a second sequence selected from the group consisting of: SEQID NO. 13; SEQ ID NO. 14; SEQ ID NO. 17; SEQ ID NO. 18; SEQ ID NO. 35;SEQ ID NO. 36; SEQ ID NO. 37; and SEQ ID NO.
 38. 20. The probe mix ofclaim 19, wherein: said first sequence is selected from the groupconsisting of: SEQ ID NO. 11; and SEQ ID NO. 25; and said secondsequence is selected from the group consisting of: SEQ ID NO. 13; SEQ IDNO. 14; SEQ ID NO. 35; and SEQ ID NO.
 36. 21. The probe mix of claim 19,wherein: said first sequence is selected from the group consisting of:SEQ ID NO. 15; and SEQ ID NO. 27; and said second sequence is selectedfrom the group consisting of: SEQ ID NO. 17; SEQ ID NO. 18; SEQ ID NO.37; and SEQ ID NO.
 38. 22. The probe mix of claim 19, wherein said atleast one helper probe includes first and second helper probes, wherein:said first sequence is SEQ ID NO. 11; said second sequence is SEQ ID NO.13 for said first helper probe; and said second sequence is SEQ ID NO.14 for said second helper probe.
 23. The probe mix of claim 19, whereinsaid at least one helper probe includes first and second helper probes,wherein: said first sequence is SEQ ID NO. 15; said second sequence isSEQ ID NO. 17 for said first helper probe; and said second sequence isSEQ ID NO. 18 for said second helper probe.
 24. A method for detectingthe presence of Neisseria meningitidis subtypes A, C and L in a sample,said method comprising the steps of: (a) contacting said sample withsaid probe of claim 1 or 17 under stringent hybridization assayconditions; and (b) detecting the presence of said probe as anindication of the presence of at least one of Neisseria meningtidissubtypes A, C and L in said sample.
 25. The method of claim 24, whereinsaid first sequence is SEQ ID NO.
 11. 26. The method of claim 24,wherein said first sequence is SEQ ID NO.
 15. 27. The method of claim24, wherein said first sequence is SEQ ID NO.
 25. 28. The method ofclaim 24, wherein said first sequence is